query: What is the result of phylogenetic analysis of SARS-CoV-2 genome sequence?

positive: In silico multi-epitope vaccine against covid19 showing effective interaction with HLA-B*15:03 The recent outbreak of severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 (SARS-CoV-2) causing coronavirus disease (covid19) has posed a great threat to human health. Previous outbreaks of SARS-CoV and Middle East respiratory Syndrome CoV (MERS-CoV) from the same CoV family had posed similar threat to human health and economic growth. To date, not even a single drug specific to any of these CoVs has been developed nor any anti-viral vaccine is available for the treatment of diseases caused by CoVs. Subunits present in spike glycoproteins of SARS-CoV and SARS-CoV-2 are involved in binding to human ACE2 Receptor which is the primary method of viral invasion. As it has been observed in the previous studies that there are very minor differences in the spike glycoproteins of SARS-CoV and SARS-CoV-2. SARS-CoV-2 has an additional furin cleavage site that makes it different from SARS-CoV (Walls et al., 2020). In this study, we have analyzed spike glycoproteins of SARS-CoV-2 and SARS-CoV phylogenetically and subjected them to selection pressure analysis. Selection pressure analysis has revealed some important sites in SARS-CoV-2 and SARS-CoV spike glycoproteins that might be involved in their pathogenicity. Further, we have developed a potential multi-epitope vaccine candidate against SARS-CoV-2 by analyzing its interactions with HLA-B*15:03 subtype. This vaccine consists of multiple T-helper (TH) cells, B-cells, and Cytotoxic T-cells (CTL) epitopes joined by linkers and an adjuvant to increase its immunogenicity. Conservation of selected epitopes in SARS, MERS, and human hosts, suggests that the designed vaccine could provide cross-protection. The vaccine is designed in silico by following a reverse vaccinology method acknowledging its antigenicity, immunogenicity, toxicity, and allergenicity. The vaccine candidate that we have designed as a result of this work shows promising result indicating its potential capability of simulating an immune response.

positive: Corona virus-An upadated review Emerging of infectious diseases such as severe acute respiratory syndrome (SARS) which is a present major menace to public health, although intense research efforts, how, when, and where new diseases are appearing which still a source of considerable uncertainty is A serious respiratory disease was recently reported in Wuhan, Hubei province, china At least 1,975cases had been reported till 25 January 2020, since the patient was hospitalized on 12 December 2019 Epidemiology research has suggested that the beginning of the virus was associated with seafood contact in Wuhan Here we considered a single patient who was one of the workers in the market and was admitted to Central Hospital of Wuhan on 26 December of 2019 He was experiencing severe respiratory syndrome which is included with dizziness and cough After analysis, it was identified as a new RNA virus strain, from the family Coronaviridae, which is generally designated as WH-Human 1’ Corona virus is also referred to 2019 –nCoV The phylogenetic analysis started that this virus was mostly closely selected to a group of SARS and MERS like corona virus these are previously been found in bats in China

positive: Analysis of RNA sequences of 3636 SARS-CoV-2 collected from 55 countries reveals selective sweep of one virus type Background & objectives: SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) is evolving with the progression of the pandemic. This study was aimed to investigate the diversity and evolution of the coronavirus SARS-CoV-2 with progression of the pandemic over time and to identify similarities and differences of viral diversity and evolution across geographical regions (countries). Methods: Publicly available data on type definitions based on whole-genome sequences of the SARS-CoV-2 sampled during December and March 2020 from 3636 infected patients spread over 55 countries were collected. Phylodynamic analyses were performed and the temporal and spatial evolution of the virus was examined. Results: It was found that (i) temporal variation in frequencies of types of the coronavirus was significant; ancestral viruses of type O were replaced by evolved viruses belonging to type A2a; (ii) spatial variation was not significant; with the spread of SARS-CoV-2, the dominant virus was the A2a type virus in every geographical region; (iii) within a geographical region, there was significant micro-level variation in the frequencies of the different viral types, and (iv) the evolved coronavirus of type A2a swept rapidly across all continents. Interpretation & conclusions: SARS-CoV-2 belonging to the A2a type possesses a non-synomymous variant (D614G) that possibly eases the entry of the virus into the lung cells of the host. This may be the reason why the A2a type has an advantage to infect and survive and as a result has rapidly swept all geographical regions. Therefore, large-scale sequencing of coronavirus genomes and, as required, of host genomes should be undertaken in India to identify regional and ethnic variation in viral composition and its interaction with host genomes. Further, careful collection of clinical and immunological data of the host can provide deep learning in relation to infection and transmission of the types of coronavirus genomes.

positive: Early phylodynamics analysis of the COVID-19 epidemics in France France was one of the first countries to be reached by the COVID-19 pandemics. Here, we analyse 196 SARS-Cov-2 genomes collected between Jan 24 and Mar 24 2020, and perform a phylodynamics analysis. In particular, we analyse the doubling time, reproduction number (Rt) and infection duration associated with the epidemic wave that was detected in incidence data starting from Feb 27. We show that a slowing down of the epidemic spread can be detected in Mar, which is consistent with the implementation of the national lock-down on Mar 17. The inferred distributions for the infection duration and Rt are in line with those estimated from contact tracing data. Overall, this analysis shows the potential to use sequence genomic data to inform public health decisions in an epidemic crisis context.

positive: Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

positive: Genetic diversity and evolution of SARS-CoV-2 Abstract COVID-19 is a viral respiratory illness caused by a new coronavirus called SARS-CoV-2. The World Health Organization declared the SARS-CoV-2 outbreak a global public health emergency. We performed genetic analyses of eighty-six complete or near-complete genomes of SARS-CoV-2 and revealed many mutations and deletions on coding and non-coding regions. These observations provided evidence of the genetic diversity and rapid evolution of this novel coronavirus.

positive: Dominant and rare SARS-Cov2 variants responsible for the COVID-19 pandemic in Athens, Greece. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel Coronavirus responsible for the Coronavirus Disease-2019 (COVID-19) pandemic. Since the beginning of the pandemic, the virus has spread in almost the entire world. Tracing and tracking virus international and local transmission has been an enormous challenge. Chains of infections starting from various countries worldwide seeded the outbreak of COVID-19 in Athens, capital city of Greece. Full-genome analysis of isolates from Athens' Hospitals and other healthcare providers revealed the variety of SARS-CoV-2 that initiated the pandemic before lock-down and passenger flight restrictions. The present work may serve as reference for resolving future lines of infection in the area and Europe especially after resumption of passenger flight connections to Athens and Greece during summer of 2020.

positive: Virus strain from a mild COVID-19 patient in Hangzhou represents a new trend in SARS-CoV-2 evolution potentially related to Furin cleavage site. The mutations in the SARS-CoV-2 virus genome during the spread of COVID-19 have been unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases, increased liver/kidney damage, and prolonged period of nuclear acid positivity, compared with patients in Wuhan, China. To investigate the underlying mechanisms, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified by gene alignment. Further phylogenetic analysis and relative synonymous codon usage heat map results suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 virus strains collected globally based on the base (C or T) at positions 8824 and 28247. ZJ01 has T at both sites and is currently the only known TT type. The prediction of the Furin cleavage site (FCS) and sequence alignment of the virus family indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was higher in the whole body, especially in glands, liver, kidneys, and colon. Thus, FCS may help SARS-CoV-2 infect these organs. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 (the source of FCS sequence-PRRA) caused mild flu-like symptoms, further showing potential in differentiating into mild COVID-19 subtypes.

positive: Importation and early local transmission of COVID-19 in Brazil, 2020 We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil.

positive: Could the D614 G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality? Increasing number of deaths due to COVID-19 pandemic has raised serious global concerns. Higher testing capacity and ample intensive care availability could explain lower mortality in some countries compared to others. Nevertheless, it is also plausible that the SARS-CoV-2 mutations giving rise to different phylogenetic clades are responsible for the obvious death disparities around the world. Current research literature linking the genetic make-up of SARS-CoV-2 with fatality is lacking. Here, we suggest that this disparity in fatality rates may be attributed to SARS-CoV-2 evolving mutations and urge the international community to begin addressing the phylogenetic clade classification of SARS-CoV-2 in relation to clinical outcomes.

positive: Analyzing hCov genome sequences: Applying Machine Intelligence and beyond Covid-19 pandemic, caused by the sars-cov-2 strain of coronavirus, has affected millions of people all over the world and taken thousands of lives. It is of utmost importance that the character of this deadly virus be studied and its nature be analysed. We present here an analysis pipeline comprising phylogenetic analysis on strains of this novel virus to track its evolutionary history among the countries uncovering several interesting relationships, followed by a classification exercise to identify the virulence of the strains and extraction of important features from its genetic material that are used subsequently to predict mutation at those interesting sites using deep learning techniques. In a nutshell, we have prepared an analysis pipeline for hCov genome sequences leveraging the power of machine intelligence and uncovered what remained apparently shrouded by raw data.

positive: Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive and GenBank repositories. Up until 27 March 2020, we downloaded 50 illumina datasets, mostly from China, USA (WA State) and Australia (VIC). A total of 30 datasets (60%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA next-generation sequencing samples. Sequencing samples from North America in GenBank (22 April 2020) present this signature with up to 39% allele frequencies among samples (n = 1,359). Australian variant signatures were more diverse than USA samples, but still, clonal events were found in these samples. Mutations in the helicase, encoded by the ORF1ab gene in SARS-CoV-2 were predominant, among others, suggesting that these regions are actively evolving. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.

positive: The Prediction of miRNAs in SARS-CoV-2 Genomes: hsa-miR Databases Identify 7 Key miRs Linked to Host Responses and Virus Pathogenicity-Related KEGG Pathways Significant for Comorbidities. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a member of the betacoronavirus family, which causes COVID-19 disease. SARS-CoV-2 pathogenicity in humans leads to increased mortality rates due to alterations of significant pathways, including some resulting in exacerbated inflammatory responses linked to the "cytokine storm" and extensive lung pathology, as well as being linked to a number of comorbidities. Our current study compared five SARS-CoV-2 sequences from different geographical regions to those from SARS, MERS and two cold viruses, OC43 and 229E, to identify the presence of miR-like sequences. We identified seven key miRs, which highlight considerable differences between the SARS-CoV-2 sequences, compared with the other viruses. The level of conservation between the five SARS-CoV-2 sequences was identical but poor compared with the other sequences, with SARS showing the highest degree of conservation. This decrease in similarity could result in reduced levels of transcriptional control, as well as a change in the physiological effect of the virus and associated host-pathogen responses. MERS and the milder symptom viruses showed greater differences and even significant sequence gaps. This divergence away from the SARS-CoV-2 sequences broadly mirrors the phylogenetic relationships obtained from the whole-genome alignments. Therefore, patterns of mutation, occurring during sequence divergence from the longer established human viruses to the more recent ones, may have led to the emergence of sequence motifs that can be related directly to the pathogenicity of SARS-CoV-2. Importantly, we identified 7 key-microRNAs (miRs 8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) with significant links to KEGG pathways linked to viral pathogenicity and host responses. According to Bioproject data (PRJNA615032), SARS-CoV-2 mediated transcriptomic alterations were similar to the target pathways of the selected 7 miRs identified in our study. This mechanism could have considerable significance in determining the symptom spectrum of future potential pandemics. KEGG pathway analysis revealed a number of critical pathways linked to the seven identified miRs that may provide insight into the interplay between the virus and comorbidities. Based on our reported findings, miRNAs may constitute potential and effective therapeutic approaches in COVID-19 and its pathological consequences.

positive: Genotypic and antigenic study of SARS-CoV-2 from an Indian isolate Coronaviruses (CoVs) are one of the largest groups of positive-sense RNA virus families within the Nidovirales order, which are further classified into four genera: alpha, beta, gamma, and delta. Coronaviruses have an extensive range of natural hosts and are known to be responsible for a broad spectrum of diseases in multiple species. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) that has unleashed a global threat to public health and the economy. Coronaviruses are extensively present in birds and mammals, with horseshoe bats (Rhinolophus affinis), being the reservoir for the ongoing SARS-CoV-2 that seems to have resulted from a zoonotic spillover to the human host, causing respiratory infections, lung injury and Acute Respiratory Distress Syndrome(ARDS). About six coronavirus serotypes are linked with the disease in humans, namely HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, and MERS-CoV. SARS-CoV-2 is the seventh CoV to infect humans. We analyzed the genome sequence of CoV-2 from isolates derived from China as well from India and encountered minute variations in their sequence. A cladogram analysis revealed the predominant strain circulating in India belongs to the A2a clad. We took one such strain (MT012098) and performed a rigorous in-silico genotypic and antigenic analysis to identify its relatedness to other strains. Further, we also performed a detailed prediction for B and T cell epitopes using BepiPred 2.0 server and NetCTL 1.2 server (DTU Bioinformatics), respectively. We hope this information may assist in an effective vaccine designing program against SARS-CoV-2.

positive: Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2 / HCoV-19) using whole genomic data The outbreak of COVID-19 started in mid-December 2019 in Wuhan, China. Up to 29 February 2020, SARS-CoV-2 (HCoV-19 / 2019-nCoV) had infected more than 85 000 people in the world. In this study, we used 93 complete genomes of SARS-CoV-2 from the GISAID EpiFlu(TM) database to investigate the evolution and human-to-human transmissions of SARS-CoV-2 in the first two months of the outbreak. We constructed haplotypes of the SARS-CoV-2 genomes, performed phylogenomic analyses and estimated the potential population size changes of the virus. The date of population expansion was calculated based on the expansion parameter tau (τ) using the formula t=τ/2u. A total of 120 substitution sites with 119 codons, including 79 non-synonymous and 40 synonymous substitutions, were found in eight coding-regions in the SARS-CoV-2 genomes. Forty non-synonymous substitutions are potentially associated with virus adaptation. No combinations were detected. The 58 haplotypes (31 found in samples from China and 31 from outside China) were identified in 93 viral genomes under study and could be classified into five groups. By applying the reported bat coronavirus genome (bat-RaTG13-CoV) as the outgroup, we found that haplotypes H13 and H38 might be considered as ancestral haplotypes, and later H1 was derived from the intermediate haplotype H3. The population size of the SARS-CoV-2 was estimated to have undergone a recent expansion on 06 January 2020, and an early expansion on 08 December 2019. Furthermore, phyloepidemiologic approaches have recovered specific directions of human-to-human transmissions and the potential sources for international infected cases.

positive: Possible Bat Origin of Severe Acute Respiratory Syndrome Coronavirus 2 We showed that severe acute respiratory syndrome coronavirus 2 is probably a novel recombinant virus. Its genome is closest to that of severe acute respiratory syndrome–related coronaviruses from horseshoe bats, and its receptor-binding domain is closest to that of pangolin viruses. Its origin and direct ancestral viruses have not been identified.

positive: A Snapshot of SARS-CoV-2 Genome Availability up to April 2020 and its Implications: Data Analysis BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been growing exponentially, affecting over 4 million people and causing enormous distress to economies and societies worldwide. A plethora of analyses based on viral sequences has already been published both in scientific journals and through non-peer-reviewed channels to investigate the genetic heterogeneity and spatiotemporal dissemination of SARS-CoV-2. However, a systematic investigation of phylogenetic information and sampling bias in the available data is lacking. Although the number of available genome sequences of SARS-CoV-2 is growing daily and the sequences show increasing phylogenetic information, country-specific data still present severe limitations and should be interpreted with caution. OBJECTIVE: The objective of this study was to determine the quality of the currently available SARS-CoV-2 full genome data in terms of sampling bias as well as phylogenetic and temporal signals to inform and guide the scientific community. METHODS: We used maximum likelihood-based methods to assess the presence of sufficient information for robust phylogenetic and phylogeographic studies in several SARS-CoV-2 sequence alignments assembled from GISAID (Global Initiative on Sharing All Influenza Data) data released between March and April 2020. RESULTS: Although the number of high-quality full genomes is growing daily, and sequence data released in April 2020 contain sufficient phylogenetic information to allow reliable inference of phylogenetic relationships, country-specific SARS-CoV-2 data sets still present severe limitations. CONCLUSIONS: At the present time, studies assessing within-country spread or transmission clusters should be considered preliminary or hypothesis-generating at best. Hence, current reports should be interpreted with caution, and concerted efforts should continue to increase the number and quality of sequences required for robust tracing of the epidemic.

positive: Comparative genomic signature representations of the emerging COVID-19 coronavirus and other coronaviruses: High identity and possible recombination between Bat and Pangolin coronaviruses Abstract Coronaviruses are responsible on respiratory diseases in animal and human. The combination of numerical encoding techniques and digital signal processing methods are becoming increasingly important in handling large genomic data. In this paper, we propose to analyze the SARS-CoV-2 genomic signature using the combination of different nucleotide representations and signal processing tools in the aim to identify its genetic origin. The sequence of SARS-CoV-2 was compared with 21 relevant sequences including bat, yak and pangolin coronavirus sequences. In addition, we developed a new algorithm to locate the nucleotide modifications. The results show that the Bat and Pangolin coronaviruses were the most related to SARS-CoV-2 with 96% and 86% of identity all along the genome. Within the S gene sequence, the Pangolin sequence presents local highest nucleotide identity. Those findings suggest genesis of SARS-Cov-2 through evolution from bat and pangolin strains. This study offers new ways to automatically characterize viruses.

positive: [Genomic analysis of a 2019-nCoV strain in the first COVID-19 patient found in Hangzhou, Zhejiang, China]. Objective: To understand the viral genomic characteristics of a 2019-nCoV strain in the first COVID-19 patient found in Hangzhou, China. Methods: Viral RNA was extracted in throat swab and sputum sample of the patient and was performed real-time reverse transcription PCR detection and obtained viral genome by high-throughput sequencing method. Phylogenetic analysis was conducted using 29 2019-nCoV genomes and 30 β-coronavirus genomes deposited in NCBI GenBank. Fifteen genomes from Wuhan were grouped by mutation sites and others were identified by Wuhan's or specific mutation sites. Results: A 29 833 bp length genome of the first 2019-nCoV strain in Hangzhou was obtained, covering full length of the coding regions of coronavirus. Phylogenetic analysis showed that the genome was closest to the genome of a bat SARS-like coronavirus strain RaTG13 with an identity of 96.11% (28 666/29 826). Among the genes between two genomes, E genes were highly conserved (99.56%), while S genes had lowest identity (92.87%), suggesting that S gene was of a faster evolutionary rate. The genome sequence similarities among 29 strains from China (Hangzhou, Wuhan, and Shenzhen), Japan, USA, and Finland, were all more than 99.9%; however, some single nucleotide polymorphisms were identified in some strains. Conclusion: The genome of Hangzhou 2019-nCoV strain was very close to the genomes of strains from other cities in China and overseas collected at early epidemic phase. The 2019-nCoV genome sequencing method used in this paper provides an useful tool for monitoring variation of viral genes.

positive: RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look. As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 'SARS-related-conserved' regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 'SARS-CoV-2-conserved-structured' regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5' UTR, frame-shifting element, and 3' UTR. Last, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 'SARS-CoV-2-conserved-unstructured' genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

positive: Genomic analysis of SARS-CoV-2 strains among Indians returning from Italy, Iran & China, & Italian tourists in India

positive: Evolutionary relationships and sequence‐structure determinants in human SARS coronavirus‐2 spike proteins for host receptor recognition Coronavirus disease 2019 (COVID‐19) is a pandemic infectious disease caused by novel Severe Acute Respiratory Syndrome coronavirus‐2 (SARS CoV‐2). The SARS CoV‐2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and SARS CoV‐2 via their glycosylated spike proteins recognize the human angiotensin converting enzyme‐2 (ACE‐2) receptor. We generated multiple sequence alignments and phylogenetic trees for representative spike proteins of SARS CoV and SARS CoV‐2 from various host sources in order to analyze the specificity in SARS CoV‐2 spike proteins required for causing infection in humans. Our results show that among the genomes analysed, two sequence regions in the N‐terminal domain (NTD); "MESEFR" and "SYLTPG" are specific to human SARS CoV‐2. In the receptor binding domain (RBD), two sequence regions; "VGGNY" and "EIYQAGSTPCNGV" and a disulfide bridge connecting 480C and 488C in the extended loop are structural determinants for the recognition of human ACE‐2 receptor. The complete genome analysis of representative SARS CoVs from bat, civet, human host sources and human SARS CoV‐2 identified the bat genome (GenBank code: MN996532.1) as closest to the recent novel human SARS CoV‐2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933 and MG772934) are evolutionary intermediates in the mutagenesis progression towards becoming human SARS CoV‐2. This article is protected by copyright. All rights reserved.

positive: Population genomics insights into the recent evolution of SARS-CoV-2 The current coronavirus disease 2019 (COVID-19) pandemic is caused by the SARS-CoV-2 virus and is still spreading rapidly worldwide. Full-genome-sequence computational analysis of the SARS-CoV-2 genome will allow us to understand the recent evolutionary events and adaptability mechanisms more accurately, as there is still neither effective therapeutic nor prophylactic strategy. In this study, we used population genetics analysis to infer the mutation rate and plausible recombination events that may have contributed to the evolution of the SARS-CoV-2 virus. Furthermore, we localized targets of recent and strong positive selection. The genomic regions that appear to be under positive selection are largely co-localized with regions in which recombination from non-human hosts appeared to have taken place in the past. Our results suggest that the pangolin coronavirus genome may have contributed to the SARS-CoV-2 genome by recombination with the bat coronavirus genome. However, we find evidence for additional recombination events that involve coronavirus genomes from other hosts, i.e., Hedgehog and Sparrow. Even though recombination events within human hosts cannot be directly assessed, due to the high similarity of SARS-CoV-2 genomes, we infer that recombinations may have recently occurred within human hosts using a linkage disequilibrium analysis. In addition, we employed an Approximate Bayesian Computation approach to estimate the parameters of a demographic scenario involving an exponential growth of the size of the SARS-CoV-2 populations that have infected European, Asian and Northern American cohorts, and we demonstrated that a rapid exponential growth in population size can support the observed polymorphism patterns in SARS-CoV-2 genomes.

positive: Full genome sequence of the first SARS-CoV-2 detected in Mexico SARS-CoV-2 was first detected in the city of Wuhan, Hubei Province, China. In this report, we describe the complete genome sequence of the first imported SARS-CoV-2, detected in a Mexican patient who had traveled to Bergamo, Italy. Phylogenetic analysis showed that this isolate belongs to subclade A2a (lineage G) and is closely related to isolates from Finland, Germany and Brazil, all of which were from patients with a history of travel to Italy. This is the first report of the complete genome sequence of this virus in Mexico.

positive: Genomic characterization and phylogenetic analysis of SARS-COV-2 in Italy This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.

positive: COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses The coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in Wuhan, China and spread around the world. Genomic analysis revealed that SARS-CoV-2 is phylogenetically related to severe acute respiratory syndrome-like (SARS-like) bat viruses, therefore bats could be the possible primary reservoir. The intermediate source of origin and transfer to humans is not known, however, the rapid human to human transfer has been confirmed widely. There is no clinically approved antiviral drug or vaccine available to be used against COVID-19. However, few broad-spectrum antiviral drugs have been evaluated against COVID-19 in clinical trials, resulted in clinical recovery. In the current review, we summarize and comparatively analyze the emergence and pathogenicity of COVID-19 infection and previous human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). We also discuss the approaches for developing effective vaccines and therapeutic combinations to cope with this viral outbreak.

positive: Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers Abstract The presence of SARS-CoV-2 in raw wastewaters has been demonstrated in many countries affected by this pandemic. Nevertheless, virus presence and infectivity in treated wastewaters, but also in the receiving water bodies is still poorly investigated. In this study, raw and treated samples from three wastewater treatment plants, and three river samples within the Milano Metropolitan Area, Italy, were surveyed for SARS-CoV-2 RNA detection by means of real time RT-PCR and infectivity test on culture cells. SARS-CoV-2 RNA was detected in raw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates). The isolated virus genome was sequenced, and belonged to the strain most spread in Europe and similar to another found in the same region. RNA presence in raw wastewater samples decreased after eight days, probably following the epidemiological trend estimated for the area. Virus infectivity was always null, indicating the natural decay of viral pathogenicity in time from emission. Samples from receiving rivers (three sites, sampled in the same dates as wastewaters) showed in some cases a positivity to real time RT-PCR, probably due to non-treated, or inefficiently treated discharges, or to the combined sewage overflows. Nevertheless, also for rivers infectivity was null. Risks for public health should be limited, although a precautionary approach to risk assessment is here advocated, giving the preliminary nature of the presented data.

positive: Emerging novel coronavirus (2019-nCoV)—current scenario, evolutionary perspective based on genome analysis and recent developments Coronaviruses are the well-known cause of severe respiratory, enteric and systemic infections in a wide range of hosts including man, mammals, fish, and avian. The scientific interest on coronaviruses increased after the emergence of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) outbreaks in 2002-2003 followed by Middle East Respiratory Syndrome CoV (MERS-CoV). This decade’s first CoV, named 2019-nCoV, emerged from Wuhan, China, and declared as ‘Public Health Emergency of International Concern’ on January 30(th), 2020 by the World Health Organization (WHO). As on February 4, 2020, 425 deaths reported in China only and one death outside China (Philippines). In a short span of time, the virus spread has been noted in 24 countries. The zoonotic transmission (animal-to-human) is suspected as the route of disease origin. The genetic analyses predict bats as the most probable source of 2019-nCoV though further investigations needed to confirm the origin of the novel virus. The ongoing nCoV outbreak highlights the hidden wild animal reservoir of the deadly viruses and possible threat of spillover zoonoses as well. The successful virus isolation attempts have made doors open for developing better diagnostics and effective vaccines helping in combating the spread of the virus to newer areas.

positive: Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS‐CoV‐2 From the beginning of 2002 and 2012, severe respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) crossed the species barriers to infect humans, causing thousands of infections and hundreds of deaths, respectively. Currently, a novel coronavirus (SARS‐CoV‐2), which has become the cause of the outbreak of Coronavirus Disease 2019 (COVID‐19), was discovered. Until 18 February 2020, there were 72 533 confirmed COVID‐19 cases (including 10 644 severe cases) and 1872 deaths in China. SARS‐CoV‐2 is spreading among the public and causing substantial burden due to its human‐to‐human transmission. However, the intermediate host of SARS‐CoV‐2 is still unclear. Finding the possible intermediate host of SARS‐CoV‐2 is imperative to prevent further spread of the epidemic. In this study, we used systematic comparison and analysis to predict the interaction between the receptor‐binding domain (RBD) of coronavirus spike protein and the host receptor, angiotensin‐converting enzyme 2 (ACE2). The interaction between the key amino acids of S protein RBD and ACE2 indicated that, other than pangolins and snakes, as previously suggested, turtles (Chrysemys picta bellii, Chelonia mydas, and Pelodiscus sinensis) may act as the potential intermediate hosts transmitting SARS‐CoV‐2 to humans.

positive: A Novel Synonymous Mutation of SARS-CoV-2: Is This Possible to Affect Their Antigenicity and Immunogenicity? The S glycoprotein of coronaviruses is important for viral entry and pathogenesis with most variable sequences. Therefore, we analyzed the S gene sequences of SARS-CoV-2 to better understand the antigenicity and immunogenicity of this virus in this study. In phylogenetic analysis, two subtypes (SARS-CoV-2a and -b) were confirmed within SARS-CoV-2 strains. These two subtypes were divided by a novel synonymous mutation of D614G. This may play a crucial role in the evolution of SARS-CoV-2 to evade the host immune system. The region containing this mutation point was confirmed as a B-cell epitope located in the S1 domain, and SARS-CoV-2b strains exhibited severe reduced antigenic indexes compared to SARS-CoV-2a in this area. This may allow these two subtypes to have different antigenicity. If the two subtypes have different serological characteristics, a vaccine for both subtypes will be more effective to prevent COVID-19. Thus, further study is urgently required to confirm the antigenicity of these two subtypes.

positive: Genomic, geographic and temporal distributions of SARS-CoV-2 mutations The COVID-19 pandemic is the most significant public health issue in recent history. Its causal agent, SARS-CoV-2, has evolved rapidly since its first emergence in December 2019. Mutations in the viral genome have critical impacts on the adaptation of viral strains to the local environment, and may alter the characteristics of viral transmission, disease manifestation, and the efficacy of treatment and vaccination. Using the complete sequences of 1,932 SARS-CoV-2 genomes, we examined the genomic, geographic and temporal distributions of aged, new, and frequent mutations of SARS-CoV-2, and identified six phylogenetic clusters of the strains, which also exhibit a geographic preference in different continents. Mutations in the form of single nucleotide variations (SNVs) provide a direct interpretation for the six phylogenetic clusters. Linkage disequilibrium, haplotype structure, evolutionary process, global distribution of mutations unveiled a sketch of the mutational history. Additionally, we found a positive correlation between the average mutation count and case fatality, and this correlation had strengthened with time, suggesting an important role of SNVs on disease outcomes. This study suggests that SNVs may become an important consideration in virus detection, clinical treatment, drug design, and vaccine development to avoid target shifting, and that continued isolation and sequencing is a crucial component in the fight against this pandemic. Significance Statement Mutation is the driving force of evolution for viruses like SARS-CoV-2, the causal agent of COVID-19. In this study, we discovered that the genome of SARS-CoV-2 is changing rapidly from the originally isolated form. These mutations have been spreading around the world and caused more than 2.5 million of infected cases and 170 thousands of deaths. We found that fourteen frequent mutations identified in this study can characterize the six main clusters of SARS-CoV-2 strains. In addition, we found the mutation burden is positively correlated with the fatality of COVID-19 patients. Understanding mutations in the SARS-CoV-2 genome will provide useful insight for the design of treatment and vaccination.

positive: Potential of large "first generation" human-to-human transmission of 2019-nCoV To investigate the genetic diversity, time origin, and evolutionary history of the 2019-nCoV outbreak in China and Thailand, a total of 12 genome sequences of the virus with known sampling date (24 December 2019 and 13 January 2020) and geographic location (primarily Wuhan city, Hubei Province, China, but also Bangkok, Thailand) were analyzed. Phylogenetic and likelihood-mapping analyses of these genome sequences were performed. On the basis of our results, the star-like signal and topology of 2019-nCoV may be indicative of potentially large "first generation" human-to-human virus transmission. We estimated that 2019-nCoV likely originated in Wuhan on 9 November 2019 (95% credible interval: 25 September 2019 and 19 December 2019), and that Wuhan is the major hub for the spread of the 2019-nCoV outbreak in China and elsewhere. Our results could be useful for designing effective prevention strategies for 2019-nCoV in China and beyond.

positive: Genomic Analysis of Early SARS-CoV-2 Variants Introduced in Mexico The COVID-19 pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to implement effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, the SARS-CoV-2 lineage A/G, containing mostly sequences from North America, and the lineage B/S containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized fourteen independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represent local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described for the country. Within this local transmission cluster, we also identified the H49Y amino acid change in the Spike protein. This mutation is a homoplasy occurring independently through time and space, and may function as a molecular marker to follow on any further spread of these viral variants throughout the country. Our results depict the general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts.IMPORTANCE Understanding the introduction, spread and establishment of SARS-CoV-2 within distinct human populations is crucial to implement effective control strategies as well as the evolution of the pandemics. In this work, we describe that the initial virus strains introduced in Mexico came from Europe and the United States and the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains having the mutation H49Y in the Spike protein, that could be further used as a molecular marker to follow viral spread within the country and the region.

positive: Understanding SARS-CoV-2: Genetic Diversity, Transmission and Cure in Human As the SARS-CoV-2 virus race around the world across the different population, there needs to be a consolidated effort to understand the divergence of demographically distributed strains. The emerging trends in SARS-CoV-2 genome data show specific mutation and genetic diversity, which could provide the basis to develop a cocktail of vaccine and may also be used to develop the region-specific diagnostic tool, thus decreasing the chances of testing failures in fields. Since the transmission of SARS-CoV-2 is subject to the extent of human interaction, the insights from the correlation of genetic diversity with epidemiological parameter would give paramount information to tackle this transmission. Previously, studies have also correlated the epidemiological data with gut microbiome and its role in immunomodulation for maintaining health status, and such information could be generated from recovered individuals from different demographic regions. It will help in designing a probiotic-based diet for modulation of the gut microbiome, and that could be another plausible prophylactic treatment option. The genomics data suggest that a specific variant of SARS-CoV-2 gets enriched with the specific demographic region. Overall, demographic data suggests that host influences mutation and expression of the virus. Hence, the experiences from the clinical intervention for that region should be considered in control and treatment strategies.

positive: A Unique Clade of SARS-CoV-2 Viruses is Associated with Lower Viral Loads in Patient Upper Airways The rapid spread of SARS-CoV-2, the causative agent of Coronavirus disease 2019 (COVID-19), has been accompanied by the emergence of distinct viral clades, though their clinical significance remains unclear. Here, we examined the genome sequences of 88 SARS-CoV-2 viruses from COVID-19 patients in Chicago, USA and identified three distinct phylogenetic clades. Clade 1 was most closely related to clades centered in New York, and showed evidence of rapid expansion across the USA, while Clade 3 was most closely related to those in Washington. Clade 2 was localized primarily to the Chicago area with limited evidence of expansion elsewhere. Average viral loads in the airways of patients infected with the rapidly spreading Clade 1 viruses were significantly higher than those of the poorly spreading Clade 2. These results show that multiple variants of SARS-CoV-2 are circulating in the USA that differ in their relative airway viral loads and potential for expansion.

positive: Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) originally arose as part of a major outbreak of respiratory disease centered on Hubei province, China. It is now a global pandemic and is a major public health concern. Taxonomically, SARS-CoV-2 was shown to be a Betacoronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2). Subsequently, betacoronaviruses from pangolins were identified as close relatives to SARS-CoV-2. Here, we perform structural modeling of the SARS-CoV-2 spike glycoprotein. Our data provide support for the similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains. We suggest this loop confers fusion activation and entry properties more in line with betacoronaviruses in lineages A and C, and be a key component in the evolution of SARS-CoV-2 with this structural loop affecting virus stability and transmission.

positive: Multi-epitope Based Peptide Vaccine Design Using Three Structural Proteins (S, E, and M) of SARS-CoV-2: An In Silico Approach Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the novel coronavirus responsible for the ongoing pandemic of coronavirus disease (COVID-19). No sustainable treatment option is available so far to tackle such a public health threat. Therefore, designing a suitable vaccine to overcome this hurdle asks for immediate attention. In this study, we targeted for a design of multi-epitope based vaccine using immunoinformatics tools. We considered the structural proteins S, E and, M of SARS-CoV-2, since they facilitate the infection of the virus into host cell and using different bioinformatics tools and servers, we predicted multiple B-cell and T-cell epitopes having potential for the required vaccine design. Phylogenetic analysis provided insight on ancestral molecular changes and molecular evolutionary relationship of S, E, and M proteins. Based on the antigenicity and surface accessibility of these proteins, eight epitopes were selected by various B cell and T cell epitope prediction tools. Molecular docking was executed to interpret the binding interactions of these epitopes and three potential epitopes WTAGAAAYY, YVYSRVKNL, and GTITVEELK were selected for their noticeable higher binding affinity scores −9.1, −7.4, and −7.0 kcal/mol, respectively. Targeted epitopes had 91.09% population coverage worldwide. In summary, we identified three epitopes having the most significant properties of designing the peptide-based vaccine against SARS-CoV-2.

positive: Molecular conservation and Differential mutation on ORF3a gene in Indian SARS-CoV2 genomes A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory proteins in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2’s virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservations of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.

positive: The first two cases of 2019-nCoV in Italy: Where they come from? A novel Coronavirus, 2019-nCoV, has been identified as the causal pathogen of an ongoing epidemic, with the first cases reported in Wuhan, China, last December 2019, and has since spread to other countries worldwide, included Europe and very recently Italy. In this short report, phylogenetic reconstruction was used to better understand the transmission dynamics of the virus from its first introduction in China focusing on the more recent evidence of infection in a couple of Chinese tourists arrived in Italy on 23rd January 2020 and labeled as Coronavirus Italian cases. A maximum clade credibility tree has been built using a dataset of 54 genome sequences of 2019-nCoV plus two closely related bat strains (SARS-like CoV) available in GenBank. Bayesian time-scaled phylogenetic analysis was implemented in BEAST 1.10.4. The Bayesian phylogenetic reconstruction showed that 2019-2020 nCoV firstly introduced in Wuhan on 25 November 2019, started epidemic transmission reaching many countries worldwide, including Europe and Italy where the two strains isolated dated back 19 January 2020, the same that the Chinese tourists arrived in Italy. Strains isolated outside China were intermixed with strains isolated in China as evidence of likely imported cases in Rome, Italy, and Europe, as well. In conclusion, this report suggests that further spread of 2019-nCoV epidemic was supported by human mobility and that quarantine of suspected or diagnosed cases is useful to prevent further transmission. Viral genome phylogenetic analysis represents a useful tool for the evaluation of transmission dynamics and preventive action.

positive: Children’s Hospital Los Angeles COVID-19 Analysis Research Database (CARD) - A Resource for Rapid SARS-CoV-2 Genome Identification Using Interactive Online Phylogenetic Tools Effective response to the Coronavirus Disease 2019 (COVID-19) pandemic requires genomic resources and bioinformatics tools for genomic epidemiology and surveillance studies that involve characterizing full-length viral genomes, identifying origins of infections, determining the relatedness of viral infections, performing phylogenetic analyses, and monitoring the continuous evolution of the SARS-CoV-2 viral genomes. The Children’s Hospital, Los Angeles (CHLA) COVID-19 Analysis Research Database (CARD) (https://covid19.cpmbiodev.net/) is a comprehensive genomic resource that provides access to full-length SARS-CoV-2 viral genomes and associated meta-data for over 30,000 (as of May 20, 2020) isolates collected from global sequencing repositories and the sequencing performed at the Center for Personalized Medicine (CPM) at CHLA. Reference phylogenetic trees of global and USA viral isolates were constructed and are periodically updated using selected high quality SARS-CoV-2 genome sequences. These provide the baseline and analytical context for identifying the origin of a viral infection, as well as the relatedness of SARS-CoV-2 genomes of interest. A web-based and interactive Phylogenetic Tree Browser supports flexible tree manipulation and advanced analysis based on keyword search while highlighting time series animation, as well as subtree export for graphical representation or offline exploration. A Virus Genome Tracker accepts complete or partial SARS-CoV-2 genome sequence, compares it against all available sequences in the database (>30,000 at time of writing), detects and annotates the variants, and places the new viral isolate within the global or USA phylogenetic contexts based upon variant profiles and haplotype comparisons, in a few seconds. The generated analysis can potentially aid in genomic surveillance to trace the transmission of any new infection. Using CHLA CARD, we demonstrate the identification of a candidate outbreak point where 13 of 31 CHLA internal isolates may have originated. We also discovered multiple indels of unknown clinical significance in the orf3a gene, and revealed a number of USA-specific variants and haplotypes.

positive: Genomic surveillance of SARS-CoV-2 reveals community transmission of a major lineage during the early pandemic phase in Brazil Despite all efforts to control the COVID-19 spread, the SARS-CoV-2 reached South America within three months after its first detection in China, and Brazil became one of the hotspots of COVID-19 in the world. Several SARS-CoV-2 lineages have been identified and some local clusters have been described in this early pandemic phase in Western countries. Here we investigated the genetic diversity of SARS-CoV-2 during the early phase (late February to late April) of the epidemic in Brazil. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 in Brazil and the community transmission of a major B.1.1 lineage defined by two amino acid substitutions in the Nucleocapsid and ORF6. This SARS-CoV-2 Brazilian lineage was probably established during February 2020 and rapidly spread through the country, reaching different Brazilian regions by the middle of March 2020. Our study also supports occasional exportations of this Brazilian B.1.1 lineage to neighboring South American countries and to more distant countries before the implementation of international air travels restrictions in Brazil.

positive: Genome analysis of SARS-CoV-2 isolates occurring in India: Present scenario Background: The origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still a debatable topic. The association of the virus spread from the market is supported by the close relation of genome sequences of environmental surface samples with virus samples from earliest patients by phylogenetic analysis. Objectives: To have an insight into the SARS-CoV-2 genome sequences reported from India for better understanding on their epidemiology and virulence. Methods: Genome sequences of Indian isolates of SARS-CoV-2 were analyzed to understand their phylogeny and divergence with respect to other isolates reported from other countries. Amino acid sequences of individual open reading frames (ORFs) from SARS-CoV-2 Indian isolates were aligned with sequences of isolates reported from other countries to identify the mutations occurred in Indian isolates. Results: Our analysis suggests that Indian SARS-CoV-2 isolates are closely related to isolates reported from other parts of the world. Most ORFs are highly conserved; mutations were also detected in some ORFs. We found that most isolates reported from India have key mutations at 614th position of the S protein and 84th position of the ORF 8, which has been reported to be associated with high virulence and high transmission rate. Conclusion: An attempt was made to understand the SARS-CoV-2 virus reported from India. SARS-CoV-2 reported from India was closely similar to other SARS-CoV-2 reported from other parts of the world, which suggests that vaccines and other therapeutic methods generated from other countries might work well in India. In addition, available sequence data suggest that majority of Indian isolates are capable of high transmission and virulence.

positive: Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.

positive: SARS-CoV-2 is sensitive to type I interferon pretreatment. SARS-CoV-2, a novel coronavirus (CoV), has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While genetically distinct from the original SARS-CoV, both group 2B CoVs share similar genome organization and origins to coronaviruses harbored in bats. Importantly, initial guidance has used insights from SARS-CoV infection to inform treatment and public health strategies. In this report, we evaluate type-I Interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication kinetics to SARS-CoV in Vero cell, the novel CoV is much more sensitive to IFN-I pretreatment. Examining transcriptional factor activation and interferon stimulated gene (ISG) induction, SARS-CoV-2 in the context of type I IFN induces phosphorylation of STAT1 and increased ISG proteins. In contrast, the original SARS-CoV has no evidence for STAT1 phosphorylation or ISG protein increases even in the presence of type I IFN pretreatment. Next, we examined IFN competent Calu3 2B4 cells finding SARS-CoV-2 had reduced viral replication relative to SARS-CoV and induced STAT1 phosphorylation late during infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonist. The absence of open reading frame (ORF) 3b and significant changes to ORF6 suggest the two key IFN antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to the IFN-I response between SARS-CoV and SARS-CoV-2. that could help inform disease progression, treatment options, and animal model development.

positive: The novel coronavirus 2019-nCoV: Its evolution and transmission into humans causing global COVID-19 pandemic A novel coronavirus strain 2019-nCoV has caused a rapid global pandemic-COVID-19. Scientists have taken onto the task of characterizing this new virus and understanding how this virus has transmitted to humans. All preliminary studies have found some striking similarities between this new virus and the SARS-CoV that caused a similar kind of epidemic in 2002–2003. Through bioinformatics tools, a great deal of information has been gathered about the origin, evolution and zoonosis of this virus. We, in this review, report the symptoms, mode of transmission and available and putative treatments to tackle 2019-nCoV infections. We also comprehensively summarize all the information so far made available regarding the genome, evolution and zoonosis of this virus.

positive: [Genomic analysis of a 2019-novel coronavirus (2019-nCoV) strain in the first COVID-19 patient found in Hangzhou] Objective: To understand the viral genomic characteristics of a 2019-novel coronavirus (2019-nCoV) strain in the first COVID-19 patient found in Hangzhou, China. Methods: Viral RNA was extracted in throat swab and sputum sample of the patient and was performed real-time reverse transcription PCR detection and obtained viral genome by high-throughput sequencing method. Phylogenetic analysis was conducted using 29 2019-nCoV genomes and 30 ß-coronavirus genomes deposited in NCBI GenBank. Fifteen genomes from Wuhan were grouped by mutation sites and others were identified by Wuhan's or specific mutation sites. Results: A 29 833 bp length genome of the first 2019-nCoV strain in Hangzhou was obtained, covering full length of the coding regions of coronavirus. Phylogenetic analysis showed that the genome was closest to the genome of a bat SARS-like coronavirus strain RaTG13 with an identity of 96.11% (28 666/29 826). Among the genes between two genomes, E genes were highly conserved (99.56%), while S genes had lowest identity (92.87%). The genome sequence similarities among 29 strains from China (Hangzhou, Wuhan, and Shenzhen), Japan, USA, and Finland, were all more than 99.9%; however, some single nucleotide polymorphisms were identified in some strains. Conclusion: The genome of Hangzhou 2019-nCoV strain was very close to the genomes of strains from other cities in China and overseas collected at early epidemic phase. The 2019-nCoV genome sequencing method used in this paper provides an useful tool for monitoring variation of viral genes.

positive: Shared SARS-CoV-2 diversity suggests localised transmission of minority variants We gratefully acknowledge the UK COVID-19 Genomics Consortium (COG UK) for funding, and Public Health Wales / Cardiff University and MRC-University of Glasgow Centre for Virus Research for making their COG-UK sequence data publicly available. COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute. The research was supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z with funding from the NIHR Oxford BRC. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. We are deeply grateful to Robert Esnouf and the BMRC Research Computing team for unfailing assistance with computational infrastructure. We also thank Benjamin Carpenter and James Docker for assistance in the laboratory, and Lorne Lonie, Maria Lopopolo, Chris Allen, John Broxholme and the WHG high-throughput genomics team for sequencing and quality control. The HIV clone p92BR025.8 was obtained through the Centre For AIDS Reagents from Drs Beatrice Hahn and Feng Gao, and the UNAIDS Virus Network (courtesy of the NIH AIDS Research and Reference Reagent Program). KAL is supported by The Wellcome Trust and The Royal Society (107652/Z/15/Z). MH, LF, MdC, GMC, NO, LAD, DB, CF and TG are supported by Li Ka Shing Foundation funding awarded to CF. PS is supported by a Wellcome Investigator Award (WT103767MA). Summary SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019 causing a global pandemic, with the United Kingdom (UK) one of the hardest hit countries. Rapid sequencing and publication of consensus genomes have enabled phylogenetic analysis of the virus, demonstrating SARS-CoV-2 evolves relatively slowly1, but with multiple sites in the genome that appear inconsistent with the overall consensus phylogeny2. To understand these discrepancies, we used veSEQ3, a targeted RNA-seq approach, to quantify minor allele frequencies in 413 clinical samples from two UK locations. We show that SARS-CoV-2 infections are characterised by extensive within-host diversity, which is frequently shared among infected individuals with patterns consistent with geographical structure. These results were reproducible in data from two other sequencing locations in the UK, where we find evidence of mixed infection by major circulating lineages with patterns that cannot readily be explained by artefacts in the data. We conclude that SARS-CoV-2 diversity is transmissible, and propose that geographic patterns are generated by transient co-circulation of distinct viral populations. Co-transmission of mixed populations could open opportunities for resolving clusters of transmission and understanding pathogenesis.

positive: Identification of the nucleotide substitutions in 62 SARS-CoV-2 sequences from Turkey A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been shown to cause coronavirus disease 2019 (COVID-19) pandemic. The first case of COVID-19 in Turkey has been declared in March 11th, 2020 and from there on, more than 150,000 people in the country have been diagnosed with the disease. In this study, 62 viral sequences from Turkey, which have been uploaded to GISAID database, were analyzed by means of their nucleotide substitutions in comparison to the reference SARS-CoV-2 genome from Wuhan. Our results indicate that the viral isolates from Turkey harbor some common mutations with the viral strains from Europe, Oceania, North America and Asia. When the mutations were evaluated, C3037T, C14408T and A23403G were found to be the most common nucleotide substitutions among the viral isolates in Turkey, which are mostly seen as linked mutations and are part of a haplotype observed high in Europe.

positive: Full-genome sequences of the first two SARS-CoV-2 viruses from India Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

positive: Computational search of hybrid human/ SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from other coronavirus strains The role of the RNAi/Dicer/Ago system to degrade RNA viruses has been elusive in mammals, which prompted authors to think that interferon (IFN) synthesis is essential in this clade relegating the RNAi defense strategy against viral infection as accessory function. We explore the theoretical possibilities that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction although this hypothesis seems counter intuitive. SARS-CoV-2 genome was therefore computational searched for exact intra pairing within the viral RNA and also hybrid exact pairing with human transcriptome over a minimum 20 bases length. Minimal segments of 20 bases length of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, among them mitochondrial deubiquitinase USP30, a subunit of ubiquitin protein ligase complex FBXO21 along with two long coding RNAs were retrieved. The hypothesis that viral originated RNAi might mediate degradation of messengers of the host transcriptome was corroborated by clinical observation and phylogenetic comparative analysis indicating a strong specificity of these hybrid pairing sequences for both SARS-CoV-2 and human genomes.

positive: Emerging phylogenetic structure of the SARS-CoV-2 pandemic Since spilling over into humans, SARS-CoV-2 has rapidly spread across the globe, accumulating significant genetic diversity. The structure of this genetic diversity, and whether it reveals epidemiological insights, are fundamental questions for understanding the evolutionary trajectory of this virus. Here we use a recently developed phylodynamic approach to uncover phylogenetic structures underlying the SARS-CoV-2 pandemic. We find support for three SARS-CoV-2 lineages co-circulating, each with significantly different demographic dynamics concordant with known epidemiological factors. For example, Lineage C emerged in Europe with a high growth rate in late February, just prior to the exponential increase in cases in several European countries. Mutations that characterize Lineage C in particular are non-synonymous and occur in functionally important gene regions responsible for viral replication and cell entry. Even though Lineages A and B had distinct demographic patterns, they were much more difficult to distinguish. Continuous application of phylogenetic approaches to track the evolutionary epidemiology of SARS-CoV-2 lineages will be increasingly important to validate the efficacy of control efforts and monitor significant evolutionary events in the future.

positive: COVID-19: The first documented coronavirus pandemic in history The novel human coronavirus disease COVID-19 has become the fifth documented pandemic since the 1918 flu pandemic. COVID-19 was first reported in Wuhan, China, and subsequently spread worldwide. The coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses based on phylogenetic analysis. SARS-CoV-2 is believed to be a spillover of an animal coronavirus and later adapted the ability of human-to-human transmission. Because the virus is highly contagious, it rapidly spreads and continuously evolves in the human population. In this review article, we discuss the basic properties, potential origin, and evolution of the novel human coronavirus. These factors may be critical for studies of pathogenicity, antiviral designs, and vaccine development against the virus.

positive: Comparative genomic analysis revealed specific mutation pattern between human coronavirus SARS-CoV-2 and Bat-SARSr-CoV RaTG13 The novel coronavirus SARS-CoV-2 (2019-nCoV) is a member of the family coronaviridae and contains a single-stranded RNA genome with positive-polarity. To reveal the evolution mechanism of SARS-CoV-2 genome, we performed comprehensive genomic analysis with newly sequenced SARS-CoV-2 strains and 20 closely related coronavirus strains. Among 98 nucleotide mutations at 93 sites of the genome among different SARS-CoV-2 strains, 58 of them caused amino acid change, indicating a result of neutral evolution. However, the ratio of nucleotide substitutions to amino acid substitutions of spike gene (9.07) between SARS-CoV-2 WIV04 and Bat-SARSr-CoV RaTG13 was extensively higher than those from comparisons between other coronaviruses (range 1.29 - 4.81). The elevated synonymous mutations between SARS-CoV-2 and RaTG13, suggesting they underwent stronger purifying selection. Moreover, their nucleotide substitutions are enriched with T:C transition, which is consistent with the mutation signature caused by deactivity of RNA 3’-to-5’ exoribonuclease (ExoN). The codon usage was similar between SARS-CoV-2 and other strains in beta-coronavirus lineage B, suggesting it had small impact on the mutation pattern. In comparison of SARS-CoV-2 WIV04 with Bat-SARSr-CoV RaTG13, the ratios of non-synonymous to synonymous substitution rates (dN/dS) was the lowest among all performed comparisons, reconfirming the evolution of SARS-CoV-2 under stringent selective pressure. Moreover, some sites of spike protein might be subjected to positive selection. Therefore, our results will help understanding the evolutionary mechanisms contribute to viral pathogenicity and its adaptation with hosts.

positive: Accommodating individual travel history, global mobility, and unsampled diversity in phylogeography: a SARS-CoV-2 case study Spatiotemporal bias in genome sequence sampling can severely confound phylogeographic inference based on discrete trait ancestral reconstruction. This has impeded our ability to accurately track the emergence and spread of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Despite the availability of unprecedented numbers of SARS-CoV-2 genomes on a global scale, evolutionary reconstructions are hindered by the slow accumulation of sequence divergence over its relatively short transmission history. When confronted with these issues, incorporating additional contextual data may critically inform phylodynamic reconstructions. Here, we present a new approach to integrate individual travel history data in Bayesian phylogeographic inference and apply it to the early spread of SARS-CoV-2, while also including global air transportation data. We demonstrate that including travel history data for each SARS-CoV-2 genome yields more realistic reconstructions of virus spread, particularly when travelers from undersampled locations are included to mitigate sampling bias. We further explore methods to ameliorate the impact of sampling bias by augmenting the phylogeographic analysis with lineages from undersampled locations in the analyses. Our reconstructions reinforce specific transmission hypotheses suggested by the inclusion of travel history data, but also suggest alternative routes of virus migration that are plausible within the epidemiological context but are not apparent with current sampling efforts. Although further research is needed to fully examine the performance of our travel-aware phylogeographic analyses with unsampled diversity and to further improve them, they represent multiple new avenues for directly addressing the colossal issue of sample bias in phylogeographic inference.

positive: Mega-phylogeny sheds light on SARS-CoV-2 spatial phylogenetic structure Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent RNA virus that spread around the planet in about 4 months. The consequences of this rapid spread on the virus evolution are under investigation. In this work, we analyzed ca. 9,000 SARS-CoV-2 genomes from Africa, America, Asia, Europe, and Oceania. We show that the virus is a complex of slightly different genetic variants that are unevenly distributed on Earth. Furthermore, we demonstrate that SARS-CoV-2 phylogeny is spatially structured. We hypothesize this could be the result of founder effects occurring as a consequence of, and local evolution occurring after, long-distance dispersal. In light of our results, we discuss how dispersal may constitute an opportunity for the virus to fix otherwise rare, and/or develop new, mutations. Based on previous studies, the possibility that this could significantly affect the virus phenotype is not remote. Relevance A cluster of pneumonia cases of unknown etiology was reported in December 2019 in Wuhan, Hubei province, China. Since then, hundreds of thousands of people have died all around the planet. Quickly after the pandemic onset, metagenomic studies showed the causative agent, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonged to the Betacoronavirus genus, responsible for spillover events in 2002 and 2012 (severe acute respiratory syndrome and Middle East respiratory syndrome, respectively). The zoonotic origins of these viruses (possibly bats, camelids, pangolins and/or palm civets) have received much attention. However, other evolutionary aspects, such as spatial variation, have received comparatively little attention. This study shows that SARS-CoV-2 variants, which we call virotypes, are heterogeneously distributed on Earth and demonstrates that the virus phylogeny is geographically structured. We explain how this may be due to founder effects combined with high mutation rates.

positive: Early Phylogenetic Diversification of SARS-CoV-2: Determination of Variants and the Effect on Epidemiology, Immunology, and Diagnostics. The phylogenetic clustering of 95 SARS-CoV-2 sequences from the first 3 months of the pandemic reveals insights into the early evolution of the virus and gives first indications of how the variants are globally distributed. Variants might become a challenge in terms of diagnostics, immunology, and effectiveness of drugs. All available whole genome sequence data from the NCBI database (March 16, 2020) were phylogenetically analyzed, and gene prediction as well as analysis of selected variants were performed. Antigenic regions and the secondary protein structure were predicted for selected variants. While some clusters are presenting the same variant with 100% identical bases, other SARS-CoV-2 lineages show a beginning diversification and phylogenetic clustering due to base substitutions and deletions in the genomes. First molecular epidemiological investigations are possible with the results by adding metadata as travelling history to the presented data. The advantage of variants in source tracing can be a challenge in terms of virulence, immune response, and immunological memory. Variants of viruses often show differences in virulence or antigenicity. This must also be considered in decisions like herd immunity. Diagnostic methods might not work if the variations or deletions are in target regions for the detection of the pathogen. One base substitution was detected in a primer binding site.

positive: Multivariate analyses of codon usage of SARS-CoV-2 and other betacoronaviruses Coronavirus disease 2019 (COVID-19) is a global health concern as it continues to spread within China and beyond. The causative agent of this disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus, which also includes severe acute respiratory syndrome-related coronavirus (SARSr-CoV) and Middle East respiratory syndrome-related coronavirus (MERSr-CoV). Codon usage of viral genes are believed to be subjected to different selection pressures in different host environments. Previous studies on codon usage of influenza A viruses helped identify viral host origins and evolution trends, however, similar studies on coronaviruses are lacking. In this study, we compared the codon usage bias using global correspondence analysis (CA), within-group CA and between-group CA. We found that the bat RaTG13 virus best matched the overall codon usage pattern of SARS-CoV-2 in orf1ab, spike and nucleocapsid genes, while the pangolin P1E virus had a more similar codon usage in membrane gene. The amino acid usage pattern of SARS-CoV-2 was generally found similar to bat and human SARSr-CoVs. However, we found greater synonymous codon usage differences between SARS-CoV-2 and its phylogenetic relatives on spike and membrane genes, suggesting these two genes of SARS-CoV-2 are subjected to different evolutionary pressures.

positive: Genetic cluster analysis of SARS-CoV-2 and the identification of those responsible for the major outbreaks in various countries A newly emerged coronavirus, SARS-CoV-2, caused severe pneumonia outbreaks in China in December 2019 and has since spread to various countries around the world. To trace the evolution route and probe the transmission dynamics of this virus, we performed phylodynamic analysis of 247 high quality genomic sequences available in the GISAID platform as of 5 March 2020. Among them, four genetic clusters, defined as super-spreaders (SSs), could be identified and were found to be responsible for the major outbreaks that subsequently occurred in various countries. SS1 was widely disseminated in Asia and the US, and mainly responsible for outbreaks in the states of Washington and California as well as South Korea, whereas SS4 contributed to the pandemic in Europe. Using the signature mutations of each SS as markers, we further analysed 1539 genome sequences reported after 29 February 2020 and found that 90% of these genomes belonged to SSs, with SS4 being the most dominant. The relative degree of contribution of each SS to the pandemic in different continents was also depicted. Identification of these super-spreaders greatly facilitates development of new strategies to control the transmission of SARS-CoV-2.

positive: Isolation and Full-Length Genome Characterization of SARS-CoV-2 from COVID-19 Cases in Northern Italy In December 2019, the novel coronavirus Severe Acquired Respiratory Syndrome SARS-CoV-2 emerged in the city of Wuhan in the Hubei province, People's Republic of China, as the etiologic agent of coronavirus disease 2019 (COVID-19), which has hence spread worldwide causing a global pandemic (1-3).….

positive: The role of phylogenetic analysis in clarifying the infection source of a COVID-19 patient

positive: Structural and Functional Implications of Spike Protein Mutational Landscape in SARS-CoV-2 SARS-CoV-2, the causative agent of COVID-19 pandemic, is an RNA virus prone to mutations. Interaction of SARS-CoV-2 Spike (S) protein with the host cell receptor, Angiotensin-I Converting Enzyme 2 (ACE2) is pivotal for attachment and entry of the virus. Yet, natural mutations acquired on S protein during the pandemic and their impact on viral infectivity, transmission dynamics and disease pathogenesis remains poorly understood. Here, we analysed 2952 SARS-CoV-2 genomes across the globe, and identified a total of 1815 non-synonymous mutations in the S-protein that fall into 54 different types. We observed that six of these distinct mutations were located in the Receptor Binding Domain (RBD) region that directly engages host ACE2. Molecular phylogenetic analysis revealed that these RBD mutations cluster into distinct phyletic clades among global subtypes of SARS-CoV-2 implying possible emergence of novel sublineages of the strain. Structure-guided homology modelling and docking analysis predicted key molecular rearrangements in the ACE2 binding interface of RBD mutants that could result in altered virus-host interactions. We propose that our findings could be significant in understanding disease dynamics and in developing vaccines, antibodies and therapeutics for COVID-19. Importance COVID-19 pandemic shows considerable variations in disease transmission and pathogenesis globally, yet reasons remain unknown. Our study identifies key S-protein mutations prevailing in SARS-CoV-2 strain that could alter viral attachment and infectivity. We propose that the interplay of these mutations could be one of the factors driving global variations in COVID-19 spread. In addition, the mutations identified in this study could be an important indicator in predicting efficacies of vaccines, antibodies and therapeutics that target SARS-CoV-2 RBD-ACE2 interface.

positive: Potential of large “first generation” human‐to‐human transmission of 2019‐nCoV To investigate the genetic diversity, time origin, and evolutionary history of the 2019‐nCoV outbreak in China and Thailand, a total of 12 genome sequences of the virus with known sampling date (24 December 2019 and 13 January 2020) and geographic location (primarily Wuhan city, Hubei Province, China, but also Bangkok, Thailand) were analyzed. Phylogenetic and likelihood‐mapping analyses of these genome sequences were performed. On the basis of our results, the star‐like signal and topology of 2019‐nCoV may be indicative of potentially large “first generation” human‐to‐human virus transmission. We estimated that 2019‐nCoV likely originated in Wuhan on 9 November 2019 (95% credible interval: 25 September 2019 and 19 December 2019), and that Wuhan is the major hub for the spread of the 2019‐nCoV outbreak in China and elsewhere. Our results could be useful for designing effective prevention strategies for 2019‐nCoV in China and beyond.

positive: A doubt of multiple introduction of SARS‐CoV‐2 in Italy: A preliminary overview The emergence of the novel betacoronavirus, recently renamed as severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has raised serious concerns due to the virus's rapid dissemination worldwide. Nevertheless, there is limited information about the genomic epidemiology of SARS‐CoV‐2 circulating in Italy from surveillance studies. The shortage of complete genomic sequences available impairs our understanding of the SARS‐CoV‐2 introduction and establishment in the country. To better understand its dynamics in Italy, we analyzed complete genomes of SARS‐CoV‐2 isolates, obtained directly from clinical samples. Our phylogenetic reconstructions suggest possible multiple introduction of SARS‐CoV‐2. Continued genomic surveillance strategies are needed to improve monitoring and understanding of the current SARS‐CoV‐2 epidemics, which might help to attenuate public health impact of infectious diseases.

positive: Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient’s sequence clustered with sequences from Germany while the tourist’s sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients’ sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

positive: Origin of Novel Coronavirus (COVID-19): A Computational Biology Study using Artificial Intelligence Origin of the COVID-19 virus has been intensely debated in the scientific community since the first infected cases were detected in December 2019. The disease has caused a global pandemic, leading to deaths of thousands of people across the world and thus finding origin of this novel coronavirus is important in responding and controlling the pandemic. Recent research results suggest that bats or pangolins might be the original hosts for the virus based on comparative studies using its genomic sequences. This paper investigates the COVID-19 virus origin by using artificial intelligence (AI) and raw genomic sequences of the virus. More than 300 genome sequences of COVID-19 infected cases collected from different countries are explored and analysed using unsupervised clustering methods. The results obtained from various AI-enabled experiments using clustering algorithms demonstrate that all examined COVID-19 virus genomes belong to a cluster that also contains bat and pangolin coronavirus genomes. This provides evidences strongly supporting scientific hypotheses that bats and pangolins are probable hosts for the COVID-19 virus. At the whole genome analysis level, our findings also indicate that bats are more likely the hosts for the COVID-19 virus than pangolins.

positive: Rapid SARS-CoV-2 whole genome sequencing for informed public health decision making in the Netherlands SARS-CoV-2 is a novel coronavirus that has rapidly spread across the globe. In the Netherlands, the first case of SARS-CoV-2 has been notified on the 27th of February. Here, we describe the first three weeks of the SARS-CoV-2 outbreak in the Netherlands, which started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also, outside the South of the Netherlands. The timely generation of whole genome sequences combined with epidemiological investigations facilitated early decision making in an attempt to control local transmission of SARS-CoV-2 in the Netherlands.

positive: About the origin of the first two Sars-CoV-2 infections in Italy: inference not supported by appropriate sequence analysis. In the 5th February 2020 issue of Journal of Medical Virology a paper was published by Giovannetti et al., entitled "The first two cases of 2019-nCoV in Italy: where they come from?"1 . In this paper a phylogenetic and evolutionary analysis was applied to the virus identified in the first two subjects diagnosed in Italy with 2019-nCoV infection, recently renamed SARS-CoV-22 , two Chinese spouses arrived in Italy for tourism. The diagnosis was performed by the virology team under direction of Maria R. Capobianchi, at the National Institute of Infectious Diseases (INMI) in Rome, Italy, where the patients are currently hospitalized. This article is protected by copyright. All rights reserved.

positive: Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2 We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.

positive: Strong evolutionary convergence of receptor-binding protein spike between COVID-19 and SARS-related coronaviruses Coronavirus Disease 2019 (COVID-19) and severe acute respiratory syndrome (SARS)-related coronaviruses (e.g., 2019-nCoV and SARS-CoV) are phylogenetically distantly related, but both are capable of infecting human hosts via the same receptor, angiotensin-converting enzyme 2, and cause similar clinical and pathological features, suggesting their phenotypic convergence. Yet, the molecular basis that underlies their phenotypic convergence remains unknown. Here, we used a recently developed molecular phyloecological approach to examine the molecular basis leading to their phenotypic convergence. Our genome-level analyses show that the spike protein, which is responsible for receptor binding, has undergone significant Darwinian selection along the branches related to 2019-nCoV and SARS-CoV. Further examination shows an unusually high proportion of evolutionary convergent amino acid sites in the receptor binding domain (RBD) of the spike protein between COVID-19 and SARS-related CoV clades, leading to the phylogenetic uniting of their RBD protein sequences. In addition to the spike protein, we also find the evolutionary convergence of its partner protein, ORF3a, suggesting their possible co-evolutionary convergence. Our results demonstrate a strong adaptive evolutionary convergence between COVID-19 and SARS-related CoV, possibly facilitating their adaptation to similar or identical receptors. Finally, it should be noted that many observed bat SARS-like CoVs that have an evolutionary convergent RBD sequence with 2019-nCoV and SARS-CoV may be pre-adapted to human host receptor ACE2, and hence would be potential new coronavirus sources to infect humans in the future.

positive: Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently co-located with TRS-B sites than expected.

positive: Isolation and rapid sharing of the 2019 novel coronavirus (SARS‐CoV‐2) from the first patient diagnosed with COVID‐19 in Australia OBJECTIVES: To describe the first isolation and sequencing of SARS‐CoV‐2 in Australia and rapid sharing of the isolate. SETTING: SARS‐CoV‐2 was isolated from a 58‐year‐old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID‐19 (the illness caused by SARS‐CoV‐2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS‐CoV‐2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS‐CoV‐2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS‐CoV‐2 genomes. Within 24 hours of isolation, the first Australian SARS‐CoV‐2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS‐CoV‐2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

positive: The Novel Coronavirus SARS‐CoV‐2: From a Zoonotic Infection to Coronavirus Disease‐19 (COVID19) OBJECTIVE: The novel coronavirus, SARS‐CoV‐2 is an international public health emergency. Until now, the intermediate host and mechanisms of interspecies jump of this virus are unknown. METHODS: Phylogenetic analysis of all available bat coronavirus (CoV) complete genomes was performed in order to analyze the relationships between bat CoV and SARS‐CoV‐2. In order to suggest a possible intermediate host, another phylogenetic reconstruction of CoV genomes obtained from animals which were hypothetically commercialized in the Chinese markets was also carried out. Moreover, mutation analysis was executed to suggest genomic regions which may have permitted the adaptation of SARS‐CoV‐2 to the human host. RESULTS: The phylogenetic analysis demonstrated that SARS‐CoV‐2 formed a cluster with the bat CoV isolate RaTG13. Possible CoV interspecies jumps among bat isolates were also observed. The phylogenetic tree reconstructed from CoV strains belonging to different animals demonstrated that SARS‐CoV‐2, bat RaTG13 and pangolin CoV genomes formed a monophyletic cluster, demonstrating that pangolins may be suggested as SARS‐CoV‐2 intermediate hosts. Three AA substitutions localized in the S1 portion of the S gene were observed, some of which have been correlated to structural modifications of the S protein which may facilitate SARS‐CoV‐2 tropism to human cells. CONCLUSIONS: Our analysis shows the tight relationship between SARS‐CoV‐2 and bat SARS‐like strains. It also hypothesizes that pangolins might have been possible intermediate hosts of the infection. Some of the observed AA substitutions in the S binding protein may serve as possible adaptation mutations in humans but more studies are needed to elucidate their function. This article is protected by copyright. All rights reserved.

positive: Genetic cluster analysis of SARS-CoV-2 and the identification of those responsible for the major outbreaks in various countries. A newly emerged coronavirus, SARS-CoV-2, caused severe pneumonia outbreaks in China in December 2019 and has since spread to various countries around the world. To trace the evolution route and probe the transmission dynamics of this virus, we performed phylodynamic analysis of 247 high quality genomic sequences available in the GISAID platform as of 5 March 2020. Among them, four genetic clusters, defined as super-spreaders (SSs), could be identified and were found to be responsible for the major outbreaks that subsequently occurred in various countries. SS1 was widely disseminated in Asia and the US, and mainly responsible for outbreaks in the states of Washington and California as well as South Korea, whereas SS4 contributed to the pandemic in Europe. Using the signature mutations of each SS as markers, we further analysed 1539 genome sequences reported after 29 February 2020 and found that 90% of these genomes belonged to SSs, with SS4 being the most dominant. The relative degree of contribution of each SS to the pandemic in different continents was also depicted. Identification of these super-spreaders greatly facilitates development of new strategies to control the transmission of SARS-CoV-2.

positive: Sequence characterization and molecular modeling of clinically relevant variants of the SARS-CoV-2 main protease The SARS-CoV-2 main protease (Mpro) is essential to viral replication and cleaves highly specific substrate sequences, making it an obvious target for inhibitor design. However, as for any virus, SARS-CoV-2 is subject to constant selection pressure, with new Mpro mutations arising over time. Identification and structural characterization of Mpro variants is thus critical for robust inhibitor design. Here we report sequence analysis, structure predictions, and molecular modeling for seventy-nine Mpro variants, constituting all clinically observed mutations in this protein as of April 29, 2020. Residue substitution is widely distributed, with some tendency toward larger and more hydrophobic residues. Modeling and protein structure network analysis suggest differences in cohesion and active site flexibility, revealing patterns in viral evolution that have relevance for drug discovery.

positive: Assessing uncertainty in the rooting of the SARS-CoV-2 phylogeny The rooting of the SARS-CoV-2 phylogeny is important for understanding the origin and early spread of the virus. Previously published phylogenies have used different rootings that do not always provide consistent results. We use several different strategies for rooting the SARS-CoV-2 tree and provide measures of statistical uncertainty for all methods. We show that methods based on the molecular clock tend to place the root in the B clade, while methods based on outgroup rooting tend to place the root in the A clade. The results from the two approaches are statistically incompatible, possibly as a consequence of deviations from a molecular clock or excess back-mutations. We also show that none of the methods provide strong statistical support for the placement of the root in any particular edge of the tree. Our results suggest that inferences on the origin and early spread of SARS-CoV-2 based on rooted trees should be interpreted with caution.

positive: Molecular characterization of SARS-CoV-2 from the first case of COVID-19 in Italy

positive: Evolutionary Trajectory for the Emergence of Novel Coronavirus SARS-CoV-2 Over the last two decades, the world experienced three outbreaks of coronaviruses with elevated morbidity rates. Currently, the global community is facing emerging virus SARS-CoV-2 belonging to Betacoronavirus, which appears to be more transmissible but less deadly than SARS-CoV. The current study aimed to track the evolutionary ancestors and different evolutionary strategies that were genetically adapted by SARS-CoV-2. Our whole-genome analysis revealed that SARS-CoV-2 was the descendant of Bat SARS/SARS-like CoVs and bats served as a natural reservoir. SARS-CoV-2 used mutations and recombination as crucial strategies in different genomic regions including the envelop, membrane, nucleocapsid, and spike glycoproteins to become a novel infectious agent. We confirmed that mutations in different genomic regions of SARS-CoV-2 have specific influence on virus reproductive adaptability, allowing for genotype adjustment and adaptations in rapidly changing environments. Moreover, for the first time we identified nine putative recombination patterns in SARS-CoV-2, which encompass spike glycoprotein, RdRp, helicase and ORF3a. Six recombination regions were spotted in the S gene and are undoubtedly important for evolutionary survival, meanwhile this permitted the virus to modify superficial antigenicity to find a way from immune reconnaissance in animals and adapt to a human host. With these combined natural selected strategies, SARS-CoV-2 emerged as a novel virus in human society.

positive: A Genomic Perspective on the Origin and Emergence of SARS-CoV-2 The ongoing pandemic of a new human coronavirus, SARS-CoV-2, has generated enormous global concern. We and others in China were involved in the initial genome sequencing of the virus. Herein, we describe what genomic data reveal about the emergence SARS-CoV-2 and discuss the gaps in our understanding of its origins.

positive: Complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in Wuhan, China Emerging and re-emerging infectious diseases, such as SARS, MERS, Zika and highly pathogenic influenza present a major threat to public health1–3. Despite intense research effort, how, when and where novel diseases appear are still the source of considerable uncertainly. A severe respiratory disease was recently reported in the city of Wuhan, Hubei province, China. At the time of writing, at least 62 suspected cases have been reported since the first patient was hospitalized on December 12nd 2019. Epidemiological investigation by the local Center for Disease Control and Prevention (CDC) suggested that the outbreak was associated with a sea food market in Wuhan. We studied seven patients who were workers at the market, and collected bronchoalveolar lavage fluid (BALF) from one patient who exhibited a severe respiratory syndrome including fever, dizziness and cough, and who was admitted to Wuhan Central Hospital on December 26th 2019. Next generation metagenomic RNA sequencing4 identified a novel RNA virus from the family Coronaviridae designed WH-Human-1 coronavirus (WHCV). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that WHCV was most closely related (89.1% nucleotide similarity similarity) to a group of Severe Acute Respiratory Syndrome (SARS)-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) previously sampled from bats in China and that have a history of genomic recombination. This outbreak highlights the ongoing capacity of viral spill-over from animals to cause severe disease in humans.

positive: Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variant sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 38 recurrent non-synonymous mutations, including ten hotspot mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed certain genotypes specific to the geographic location. We also found co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets We have also created an inclusive unified database (http://genoma.ma/covid-19/) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.

positive: Reply to Sánchez-Pacheco et al., Chookajorn, and Mavian et al.: Explaining phylogenetic network analysis of SARS-CoV-2 genomes

positive: Snapshot of the evolution and mutation patterns of SARS-CoV-2 The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.

positive: Are pangolins the intermediate host of the 2019 novel coronavirus (2019-nCoV) ? The outbreak of 2019-nCoV pneumonia (COVID-19) in the city of Wuhan, China has resulted in more than 70,000 laboratory confirmed cases, and recent studies showed that 2019-nCoV (SARS-CoV-2) could be of bat origin but involve other potential intermediate hosts. In this study, we assembled the genomes of coronaviruses identified in sick pangolins. The molecular and phylogenetic analyses showed that pangolin Coronaviruses (pangolin-CoV) are genetically related to both the 2019-nCoV and bat Coronaviruses but do not support the 2019-nCoV arose directly from the pangolin-CoV. Our study also suggested that pangolin be natural host of Betacoronavirus, with a potential to infect humans. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. Conservation of wildlife and limits of the exposures of humans to wildlife will be important to minimize the spillover risks of coronaviruses from wild animals to humans.

positive: The novel coronavirus SARS-CoV-2: From a zoonotic infection to coronavirus disease 2019 The novel coronavirus (CoV), severe acute respiratory syndrome (SARS)-CoV-2 is an international public health emergency. Until now, the intermediate host and mechanisms of the interspecies jump of this virus are unknown. Phylogenetic analysis of all available bat CoV complete genomes was performed to analyze the relationships between bat CoV and SARS-CoV-2. To suggest a possible intermediate host, another phylogenetic reconstruction of CoV genomes obtained from animals that were hypothetically commercialized in the Chinese markets was also carried out. Moreover, mutation analysis was executed to suggest genomic regions that may have permitted the adaptation of SARS-CoV-2 to the human host. The phylogenetic analysis demonstrated that SARS-CoV-2 formed a cluster with the bat CoV isolate RaTG13. Possible CoV interspecies jumps among bat isolates were also observed. The phylogenetic tree reconstructed from CoV strains belonging to different animals demonstrated that SARS-CoV-2, bat RaTG13, and pangolin CoV genomes formed a monophyletic cluster, demonstrating that pangolins may be suggested as SARS-CoV-2 intermediate hosts. Three AA substitutions localized in the S1 portion of the S gene were observed, some of which have been correlated to structural modifications of the S protein which may facilitate SARS-CoV-2 tropism to human cells. Our analysis shows the tight relationship between SARS-CoV-2 and bat SARS-like strains. It also hypothesizes that pangolins might have been possible intermediate hosts of the infection. Some of the observed AA substitutions in the S-binding protein may serve as possible adaptation mutations in humans but more studies are needed to elucidate their function.

positive: Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China Summary Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China’s most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province’s large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

positive: Analysis of SARS-CoV-2 RNA-dependent RNA polymerase as a potential therapeutic drug target using a computational approach BACKGROUND: The Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) outbreak originating in Wuhan, China, has raised global health concerns and the pandemic has now been reported on all inhabited continents. Hitherto, no antiviral drug is available to combat this viral outbreak. METHODS: Keeping in mind the urgency of the situation, the current study was designed to devise new strategies for drug discovery and/or repositioning against SARS-CoV-2. In the current study, RNA-dependent RNA polymerase (RdRp), which regulates viral replication, is proposed as a potential therapeutic target to inhibit viral infection. RESULTS: Evolutionary studies of whole-genome sequences of SARS-CoV-2 represent high similarity (> 90%) with other SARS viruses. Targeting the RdRp active sites, ASP760 and ASP761, by antiviral drugs could be a potential therapeutic option for inhibition of coronavirus RdRp, and thus viral replication. Target-based virtual screening and molecular docking results show that the antiviral Galidesivir and its structurally similar compounds have shown promise against SARS-CoV-2. CONCLUSIONS: The anti-polymerase drugs predicted here—CID123624208 and CID11687749—may be considered for in vitro and in vivo clinical trials.

positive: Viral Phylogenomics Using an Alignment-Free Method: A Three-Step Approach to Determine Optimal Length of k-mer The development of rapid, economical genome sequencing has shed new light on the classification of viruses. As of October 2016, the National Center for Biotechnology Information (NCBI) database contained >2 million viral genome sequences and a reference set of ~4000 viral genome sequences that cover a wide range of known viral families. Whole-genome sequences can be used to improve viral classification and provide insight into the viral “tree of life”. However, due to the lack of evolutionary conservation amongst diverse viruses, it is not feasible to build a viral tree of life using traditional phylogenetic methods based on conserved proteins. In this study, we used an alignment-free method that uses k-mers as genomic features for a large-scale comparison of complete viral genomes available in RefSeq. To determine the optimal feature length, k (an essential step in constructing a meaningful dendrogram), we designed a comprehensive strategy that combines three approaches: (1) cumulative relative entropy, (2) average number of common features among genomes, and (3) the Shannon diversity index. This strategy was used to determine k for all 3,905 complete viral genomes in RefSeq. The resulting dendrogram shows consistency with the viral taxonomy of the ICTV and the Baltimore classification of viruses.

positive: Phylogenetic analysis of the first four SARS-CoV-2 cases in Chile The current pandemic caused by the new coronavirus is a worldwide public health concern. To aboard this emergency, and like never before, scientific groups around the world have been working in a fast and coordinated way to get the maximum of information about this virus when it has been almost 3 months since the first cases were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub-classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection.

positive: Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? The outbreak of a novel corona Virus Disease 2019 (COVID-19) in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans.

positive: A new coronavirus associated with human respiratory disease in China Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health1-3. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China5. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

positive: Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report BACKGROUND: The emergence of a novel coronavirus, SARS-CoV-2, in December 2019, progressed to become a world pandemic in a few months and reached South Africa at the beginning of March. To investigate introduction and understand the early transmission dynamics of the virus, we formed the South African Network for Genomics Surveillance of COVID (SANGS_COVID), a network of ten government and university laboratories. Here, we present the first results of this effort, which is a molecular epidemiological study of the first twenty-one SARS-CoV-2 whole genomes sampled in the first port of entry, KwaZulu-Natal (KZN), during the first month of the epidemic. By combining this with calculations of the effective reproduction number (R), we aim to shed light on the patterns of infections that define the epidemic in South Africa. METHODS: R was calculated using positive cases and deaths from reports provided by the four major provinces. Molecular epidemiology investigation involved sequencing viral genomes from patients in KZN using ARCTIC protocols and assembling whole genomes using meticulous alignment methods. Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian trees, lineage classification and molecular clock calculations. FINDINGS: The epidemic in South Africa has been very heterogeneous. Two of the largest provinces, Gauteng, home of the two large metropolis Johannesburg and Pretoria, and KwaZulu-Natal, home of the third largest city in the country Durban, had a slow growth rate on the number of detected cases. Whereas, Western Cape, home of Cape Town, and the Eastern Cape provinces the epidemic is spreading fast. Our estimates of transmission potential for South Africa suggest a decreasing transmission potential towards R=1 since the first cases and deaths have been reported. However, between 06 May and 18 May 2020, we estimate that R was on average 1.39 (1.04 – 2.15, 95% CI). We also demonstrate that early transmission in KZN, and most probably in all main regions of SA, was associated with multiple international introductions and dominated by lineages B1 and B. The study also provides evidence for locally acquired infections in a hospital in Durban within the first month of the epidemic, which inflated early mortality in KZN. INTERPRETATION: This first report of SANGS_COVID consortium focuses on understanding the epidemic heterogeneity and introduction of SARS-CoV-2 strains in the first month of the epidemic in South Africa. The early introduction of SARS-CoV-2 in KZN included caused a localized outbreak in a hospital, provides potential explanations for the initially high death rates in the province. The current high rate of transmission of COVID-19 in the Western Cape and Eastern Cape highlights the crucial need to strength local genomic surveillance in South Africa. FUNDING: UKZN Flagship Program entitled: Afrocentric Precision Approach to Control Health Epidemic, by a research Flagship grant from the South African Medical Research Council (MRC-RFA-UFSP-01-2013/UKZN HIVEPI, by the the Technology Innovation Agency and the the Department of Science and Innovation and by National Human Genome Re- search Institute of the National Institutes of Health under Award Number U24HG006941. H3ABioNet is an initiative of the Human Health and Heredity in Africa Consortium (H3Africa).

positive: Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia OBJECTIVES: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. SETTING: SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

positive: Novel SARS-CoV-2/COVID-19: Origin, pathogenesis, genes and genetic variations, immune responses and phylogenetic analysis In this review, we focused on the origins of the novel coronavirus (SARS-CoV-2), origin, pathogenesis, immune responses, genes and genetic variations, phylogenetic analyses, and potential therapeutic strategies to summarize approaches for developing broadly effective preventions and vaccines to cope COVID-19. Towards the end of 2019, SARS-CoV-2 has emerged in association with the SARS, later was named COVID-19 caused an environment of chaos worldwide and infected a massive number of lives. Since these epidemics or pandemics had spread to 210 countries and territories around the world and 2 international conveyances with 6,467,229 confirmed cases, including, 382,766 deaths, as of June 03, 2020 (https://www.worldometers.info/coronavirus/), hence the World Health Organization declared it as a global Public Health Emergency. There are no clinically approved vaccines or antiviral drugs available for either of new or old corona infections; thus, the development of effective therapeutic and preventive strategies that can be readily available to cope with these strains.

positive: Epitope-Based Peptide Vaccine Against Severe Acute Respiratory Syndrome-Coronavirus-2 Nucleocapsid Protein: An in silico Approach With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. SARS-CoV-2 is a member of the Coronaviridae family, which is transmitted from animal to human and because of being contagious, further it transmitted human to human. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019) as a global pandemic. But, no specific medications are available for the treatment of COVID-19 so far. As a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication as well as interferes with host immune responses. We have comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we have predicted the most immunogenic epitope for T-cell as well as B-cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates.

positive: Broad Host Range of SARS-CoV-2 Predicted by Comparative and Structural Analysis of ACE2 in Vertebrates The novel coronavirus SARS-CoV-2 is the cause of Coronavirus Disease-2019 (COVID-19). The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of 410 vertebrates, including 252 mammals, to study cross-species conservation of ACE2 and its likelihood to function as a SARS-CoV-2 receptor. We designed a five-category ranking score based on the conservation properties of 25 amino acids important for the binding between receptor and virus, classifying all species from very high to very low. Only mammals fell into the medium to very high categories, and only catarrhine primates in the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 binding, and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (<0.1%) variants in 10/25 binding sites. In addition, we observed evidence of positive selection in ACE2 in multiple species, including bats. Utilized appropriately, our results may lead to the identification of intermediate host species for SARS-CoV-2, justify the selection of animal models of COVID-19, and assist the conservation of animals both in native habitats and in human care.

positive: Sequence variation among SARS-CoV-2 isolates in Taiwan Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, followed by those from other countries starting in early March. Additionally, several cases could not be traced to any imported cases and were suspected as sporadic local transmission. Twelve full viral genomes were determined in this study by Illumina sequencing either from virus isolates or directly from specimens, among which 5 originated from clustered infections. Phylogenetic tree analysis revealed that these sequences were in different clades, indicating that no major strain has been circulating in Taiwan. A deletion in open reading frame 8 was found in one isolate. Only a 4-nucleotide difference was observed among the 5 genomes from clustered infections.

positive: Virus Isolation from the First Patient with SARS-CoV-2 in Korea Novel coronavirus (SARS-CoV-2) is found to cause a large outbreak started from Wuhan since December 2019 in China and SARS-CoV-2 infections have been reported with epidemiological linkage to China in 25 countries until now. We isolated SARS-CoV-2 from the oropharyngeal sample obtained from the patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea. Cytopathic effects of SARS-CoV-2 in the Vero cell cultures were confluent 3 days after the first blind passage of the sample. Coronavirus was confirmed with spherical particle having a fringe reminiscent of crown on transmission electron microscopy. Phylogenetic analyses of whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan.

positive: Sampling bias and incorrect rooting make phylogenetic network tracing of SARS-COV-2 infections unreliable

positive: Population Genetics of SARS-CoV-2: Disentangling Effects of Sampling Bias and Infection Clusters A novel RNA virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the ongoing outbreak of coronavirus disease 2019 (COVID-19). Population genetic analysis could be useful for investigating the origin and evolutionary dynamics of COVID-19. However, due to extensive sampling bias and existence of infection clusters during the epidemic spread, direct applications of existing approaches can lead to biased parameter estimations and data misinterpretation. In this study, we first present robust estimator for the time to the most recent common ancestor (TMRCA) and the mutation rate, and then apply the approach to analyze 12,909 genomic sequences of SARS-CoV-2. The mutation rate is inferred to be 8.69 × 10(-4) per site per year with a 95% confidence interval (CI) of [8.61 × 10(-4), 8.77 × 10(-4)], and the TMRCA of the samples inferred to be Nov 28, 2019 with a 95% CI of [Oct 20, 2019, Dec 9, 2019]. The results indicate that COVID-19 might originate earlier than and outside of Wuhan Seafood Market. We further demonstrate that genetic polymorphism patterns, including the enrichment of specific haplotypes and the temporal allele frequency trajectories generated from infection clusters, are similar to those caused by evolutionary forces such as natural selection. Our results show that population genetic methods need to be developed to efficiently detangle the effects of sampling bias and infection clusters to gain insights into the evolutionary mechanism of SARS-CoV-2. Software for implementing VirusMuT can be downloaded at https://bigd.big.ac.cn/biocode/tools/BT007081.

positive: Properties of Coronavirus and SARS-CoV-2. were identified beginning with the discovery of SARS-CoV in 2002. With the recent detection of SARS-CoV-2, there are now seven human coronaviruses. Those that cause mild diseases are the 229E, OC43, NL63 and HKU1, and the pathogenic species are SARS-CoV, MERS-CoV and SARS-CoV-2 Coronaviruses (order Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae) are spherical (125nm diameter), and enveloped with club-shaped spikes on the surface giving the appearance of a solar corona. Within the helically symmetrical nucleocapsid is the large positive sense, single stranded RNA. Of the four coronavirus genera (α,β,γ,δ), human coronaviruses (HCoVs) are classified under α-CoV (HCoV-229E and NL63) and β-CoV (MERS-CoV, SARS-CoV, HCoVOC43 and HCoV-HKU1). SARS-CoV-2 is a β-CoV and shows fairly close relatedness with two bat-derived CoV-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21. Even so, its genome is similar to that of the typical CoVs. SARS-CoV and MERS-CoV originated in bats, and it appears to be so for SARS-CoV-2 as well. The possibility of an intermediate host facilitating the emergence of the virus in humans has already been shown with civet cats acting as intermediate hosts for SARS-CoVs, and dromedary camels for MERS-CoV. Human-to-human transmission is primarily achieved through close contact of respiratory droplets, direct contact with the infected individuals, or by contact with contaminated objects and surfaces. The coronaviral genome contains four major structural proteins: the spike (S), membrane (M), envelope (E) and the nucleocapsid (N) protein, all of which are encoded within the 3' end of the genome. The S protein mediates attachment of the virus to the host cell surface receptors resulting in fusion and subsequent viral entry. The M protein is the most abundant protein and defines the shape of the viral envelope. The E protein is the smallest of the major structural proteins and participates in viral assembly and budding. The N protein is the only one that binds to the RNA genome and is also involved in viral assembly and budding. Replication of coronaviruses begin with attachment and entry. Attachment of the virus to the host cell is initiated by interactions between the S protein and its specific receptor. Following receptor binding, the virus enters host cell cytosol via cleavage of S protein by a protease enzyme, followed by fusion of the viral and cellular membranes. The next step is the translation of the replicase gene from the virion genomic RNA and then translation and assembly of the viral replicase complexes. Following replication and subgenomic RNA synthesis, encapsidation occurs resulting in the formation of the mature virus. Following assembly, virions are transported to the cell surface in vesicles and released by exocytosis.

positive: Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV After the outbreak of the severe acute respiratory syndrome (SARS) in the world in 2003, human coronaviruses (HCoVs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. Recently, a new emerged HCoV isolated from the respiratory epithelium of unexplained pneumonia patients in the Wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus causes acute lung symptoms, leading to a condition that has been named as “coronavirus disease 2019” (COVID-19). The emergence of SARS-CoV-2 and of SARS-CoV caused widespread fear and concern and has threatened global health security. There are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. The goal of this work is to systematically review and compare between SARS-CoV and SARS-CoV-2 in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms.

positive: Molecular evolution and phylogenetic analysis of SARS-CoV-2 and hosts ACE2 protein suggest Malayan pangolin as intermediary host An emergence of a novel coronavirus, causative agent of COVID19, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), occurred due to cross-species transmission. Coronaviruses are a large family of viruses able to infect a great number of hosts. Entrance of SARS-CoV-2 depends on the surface (S) protein interaction with host ACE2 protein and cleavage by TMPRSS2. ACE2 could be a species-specific barrier that interferes with bat-to-human coronavirus cross-species transmission. Molecular analysis supported bats as natural hosts for SARS-CoV and involved them in MERS-CoV origin. The genomic similarity between bat RaTG13 CoV strain and SARS-CoV-2 implicates bats in the origin of the new outbreak. Additionally, there is a hypothesis for the zoonotic transmission based on contact with Malayan pangolins by humans in Huanan seafood market in Wuhan, China. To investigate bats and pangolin as hosts in SARS-CoV-2 cross-species transmission, we perform an evolutionary analysis combining viral and host phylogenies and divergence of ACE2 and TMPRSS2 amino acid sequences between CoV hosts. Phylogeny showed SARS-like-CoV-2 strains that infected pangolin and bats are close to SARS-CoV-2. In contrast to TMPRSS2, pangolin ACE2 amino acid sequence has low evolutionary divergence compared with humans and is more divergent from bats. Comparing SARS-CoV with SARS-CoV-2 origins, pangolin has yet lower ACE2 evolutionary divergence with humans than civet-the main intermediary host of SARS-CoV. Thus, pangolin has become an opportune host to intermediates bat-to-human SARS-CoV-2 jump and entry.

positive: [Genomic characterization of SARS coronavirus: a novel member of coronavirus]. In March 2003, SARS-CoV, a novel coronavirus which has been proved to be a pathogen causing Severe Acute Respiratory Syndrome (SARS). The complete genome of SARS-CoV has been sequenced by international collaboration including China. In the present study, the genome sequences were collected from NCBI and genomic characterization was analyzed. SARS-CoV has a genome of 28-30 kb including 11 ORFs (Open Reading Frames), which is consistent with that of coronavirus family, and its genome organization is similar to those of other coronaviruses as well. SARS-CoV evolutionally closes to other coronavirus in their corresponding proteins, such as spike protein, small membrane protein and nucleocapsid protein. In some regions of the genome, the genomic sequence of SARS-CoV was significantly different from that of other coronavirus, and has a self-conservative genomic sequence. Moreover, its encoding protein sequences were greatly different from those of other coronavirus. The analysis indicated that SARS-CoV has lower redundancy, that is, it has a high variation possibility. It may not be a variant of other coronaviruses but a novel coronavirus, which existed independently in nature and was not recognized by human being before, although SARS-CoV is morphologically similar to other coronavirus and belongs to coronavirus family. The sequences of its genes and encoding proteins are substantially different from those of other coronavirus.

positive: Genomic surveillance reveals multiple introductions of SARS-CoV-2 into Northern California The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, with >52,000 cases in California as of May 4, 2020. Here we investigate the genomic epidemiology of SARS-CoV-2 in Northern California from late January to mid-March 2020, using samples from 36 patients spanning 9 counties and the Grand Princess cruise ship. Phylogenetic analyses revealed the cryptic introduction of at least 7 different SARS-CoV-2 lineages into California, including epidemic WA1 strains associated with Washington State, with lack of a predominant lineage and limited transmission between communities. Lineages associated with outbreak clusters in 2 counties were defined by a single base substitution in the viral genome. These findings support contact tracing, social distancing, and travel restrictions to contain SARS-CoV-2 spread in California and other states.

positive: Phylogenetic Analysis of SARS-CoV-2 Genomes in Turkey COVID-19 has effectively spread worldwide. As of May 2020, Turkey is among the top ten countries with the most cases. A comprehensive genomic characterization of the virus isolates in Turkey is yet to be carried out. Here, we built a phylogenetic tree with globally obtained 15,277 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. We identified the subtypes based on the phylogenetic clustering in comparison with the previously annotated classifications. We performed a phylogenetic analysis of the first thirty SARS-CoV-2 genomes isolated and sequenced in Turkey. We suggest that the first introduction of the virus to the country is earlier than the first reported case of infection. Virus genomes isolated from Turkey are dispersed among most types in the phylogenetic tree. We find two of the seventeen sub-clusters enriched with the isolates of Turkey, which likely have spread expansively in the country. Finally, we traced virus genomes based on their phylogenetic placements. This analysis suggested multiple independent international introductions of the virus and revealed a hub for the inland transmission. We released a web application to track the global and interprovincial virus spread of the isolates from Turkey in comparison to thousands of genomes worldwide.

positive: Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses However, the external subdomain of Spike's receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses Remarkably, its orf3b encodes a completely novel short protein Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection FAU - Chan, Jasper Fuk-Woo

positive: Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Egypt This report announces the complete genome sequences of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates detected in Egypt. The isolates were obtained from oropharyngeal swab specimens from two Egyptians in Upper and Lower Egypt. Sequence analysis showed mutations that differentiate Egyptian strains from the reference strain 2019-nCoV WHU01.

positive: Molecular tracing of SARS-CoV-2 in Italy in the first three months of the epidemic The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

positive: Viral Diversity of Microbats within the South West Botanical Province of Western Australia Bats are known reservoirs of a wide variety of viruses that rarely result in overt clinical disease in the bat host. However, anthropogenic influences on the landscape and climate can change species assemblages and interactions, as well as undermine host-resilience. The cumulative result is a disturbance of bat–pathogen dynamics, which facilitate spillover events to sympatric species, and may threaten bat communities already facing synergistic stressors through ecological change. Therefore, characterisation of viral pathogens in bat communities provides important basal information to monitor and predict the emergence of diseases relevant to conservation and public health. This study used targeted molecular techniques, serological assays and next generation sequencing to characterise adenoviruses, coronaviruses and paramyxoviruses from 11 species of insectivorous bats within the South West Botanical Province of Western Australia. Phylogenetic analysis indicated complex ecological interactions including virus–host associations, cross-species infections, and multiple viral strains circulating concurrently within selected bat populations. Additionally, we describe the entire coding sequences for five alphacoronaviruses (representing four putative new species), and one novel adenovirus. Results indicate that viral burden (both prevalence and richness) is not homogeneous among species, with Chalinolobus gouldii identified as a key epidemiological element within the studied communities.

positive: Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus Recently, a novel coronavirus (2019-nCoV) has emerged from Wuhan, China, causing symptoms in humans similar to those caused by severe acute respiratory syndrome coronavirus (SARS-CoV). Since the SARS-CoV outbreak in 2002, extensive structural analyses have revealed key atomic-level interactions between the SARS-CoV spike protein receptor-binding domain (RBD) and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. Here, we analyzed the potential receptor usage by 2019-nCoV, based on the rich knowledge about SARS-CoV and the newly released sequence of 2019-nCoV. First, the sequence of 2019-nCoV RBD, including its receptor-binding motif (RBM) that directly contacts ACE2, is similar to that of SARS-CoV, strongly suggesting that 2019-nCoV uses ACE2 as its receptor. Second, several critical residues in 2019-nCoV RBM (particularly Gln493) provide favorable interactions with human ACE2, consistent with 2019-nCoV’s capacity for human cell infection. Third, several other critical residues in 2019-nCoV RBM (particularly Asn501) are compatible with, but not ideal for, binding human ACE2, suggesting that 2019-nCoV has acquired some capacity for human-to-human transmission. Last, while phylogenetic analysis indicates a bat origin of 2019-nCoV, 2019-nCoV also potentially recognizes ACE2 from a diversity of animal species (except mice and rats), implicating these animal species as possible intermediate hosts or animal models for 2019-nCoV infections. These analyses provide insights into the receptor usage, cell entry, host cell infectivity and animal origin of 2019-nCoV and may help epidemic surveillance and preventive measures against 2019-nCoV. IMPORTANCE The recent emergence of Wuhan coronavirus (2019-nCoV) puts the world on alert. 2019-nCoV is reminiscent of the SARS-CoV outbreak in 2002 to 2003. Our decade-long structural studies on the receptor recognition by SARS-CoV have identified key interactions between SARS-CoV spike protein and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. One of the goals of SARS-CoV research was to build an atomic-level iterative framework of virus-receptor interactions to facilitate epidemic surveillance, predict species-specific receptor usage, and identify potential animal hosts and animal models of viruses. Based on the sequence of 2019-nCoV spike protein, we apply this predictive framework to provide novel insights into the receptor usage and likely host range of 2019-nCoV. This study provides a robust test of this reiterative framework, providing the basic, translational, and public health research communities with predictive insights that may help study and battle this novel 2019-nCoV.

positive: Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has a capability of gaining rapid mutations as the virus spreads. Whole genome sequencing data offers a wide range of opportunities to study the mutation dynamics. The advantage of increasing amount of whole genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity and to investigate the implications of those mutations in protein stability and viral transmission. Four proteins, surface glycoprotein, nucleocapsid, ORF1ab and ORF8 showed frequent mutations, while envelop, membrane, ORF6 and ORF7a proteins showed conservation in terms of amino acid substitutions. Some of the mutations across different proteins showed co-occurrence, suggesting their functional cooperation in stability, transmission and adaptability. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Analysis of protein structure stability of surface glycoprotein mutants indicated viability of specific variants and are more prone to be temporally and spatially distributed across the globe. Similar empirical analysis of other proteins indicated existence of important functional implications of several variants. Analysis of co-occurred mutants indicated their structural and/or functional interaction among different SARS-COV-2 proteins. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing and containment of the disease.

positive: Genetic analysis of SARS-CoV-2 strains collected from North Africa: viral origins and mutational spectrum In Morocco two waves of SARS-CoV-2 infections have been recorded. The first one occurred from March 02, 2020 with infections mostly imported from Europe and the second one dominated by local infections. At the time of writing, the genetic diversity of Moroccan isolates of SARS-CoV-2 has not yet been reported. The present study aimed to analyze first the genomic variation of the twenty-eight Moroccan strains of SARS-CoV-2 isolated from March 03, 2020 to May 15, 2020, to compare their distributions with twelve other viral genomes from North Africa as well as to identify their possible sources. Our finding revealed 61 mutations in the Moroccan genomes of SARS-CoV-2 compared to the reference sequence Wuhan-Hu-1/2019, of them 23 (37.7%) were present in two or more genomes. Focusing on non-synonymous mutations, 29 (47.54%) were distributed in five genes (ORF1ab, spike, membrane, nucleocapsid and ORF3a) with variable frequencies. The non-structural protein coding regions nsp3-Multi domain and nsp12-RdRp of the ORF1ab gene harbored more mutations, with six for each. The comparison of genetic variants of fourty North African strains revealed that two non-synonymous mutations D614G (in spike) and Q57H (in ORF3a) were common in four countries (Morocco, Tunisia, Algeria and Egypt), with a prevalence of 92.5% (n = 37) and 42.5% (n = 17), respectively, of the total genomes. Phylogenetic analysis showed that the Moroccan and Tunisian SARS-CoV-2 strains were closely related to those from different origins (Asia, Europe, North and South America) and distributed in different distinct subclades. This could indicate different sources of infection with no specific strain dominating yet in in these countries. These results have the potential to lead to new comprehensive investigations combining genomic data, epidemiological information and the clinical characteristics of patients with SARS-CoV-2.

positive: Origin and cross-species transmission of bat coronaviruses in China Bats are presumed reservoirs of diverse coronaviruses (CoVs) including progenitors of Severe Acute Respiratory Syndrome (SARS)-CoV and SARS-CoV-2, the causative agent of COVID-19. However, the evolution and diversification of these coronaviruses remains poorly understood. We used a Bayesian statistical framework and sequence data from all known bat-CoVs (including 630 novel CoV sequences) to study their macroevolution, cross-species transmission, and dispersal in China. We find that host-switching was more frequent and across more distantly related host taxa in alpha-than beta-CoVs, and more highly constrained by phylogenetic distance for beta-CoVs. We show that inter-family and -genus switching is most common in Rhinolophidae and the genus Rhinolophus. Our analyses identify the host taxa and geographic regions that define hotspots of CoV evolutionary diversity in China that could help target bat-CoV discovery for proactive zoonotic disease surveillance. Finally, we present a phylogenetic analysis suggesting a likely origin for SARS-CoV-2 in Rhinolophus spp. bats.

positive: Phylogenomics and phylodynamics of SARS-CoV-2 retrieved genomes from India The ongoing SARS-CoV-2 pandemic is one of the biggest outbreaks after the Spanish flu of 1918. Understanding the epidemiology of viral outbreaks is the first step towards vaccine development programs. This is the first phylodynamics study attempted on of SARS-CoV-2 genomes from India to infer its current evolution in the context of an ongoing pandemic. Out of 286 retrieved SARS-CoV-2 whole genomes from India, 138 haplotypes were generated and analyzed. Median-joining network was built to investigate the relatedness of SARS-CoV-2 haplotypes in India. The BDSIR package of BEAST2 was used to calculate the reproduction number (R0) and the infectious rate of the virus. Past and current population trend was investigated using the stamp date method in coalescent Bayesian skyline plot, implemented in BEAST2 and by exponential growth prior in BEAST 1.10.4. Median-joining network reveals two distinct ancestral clusters A and B showing genetic affinities with Wuhan outbreak sample. The network also illustrates the autochthonous development of isolates in a few instances. High basic reproduction number of SARS-nCoV-2 in India points towards the phase of active community transmission. The Bayesian skyline plot revel exponential rise in the effective population size (Ne) of Indian isolates from the first week of January to the first week of April 2020. More genome sequencing and analyses of the virus will be required in coming days to monitor COVID19 after the upliftment of lock down in India.

positive: Phylogenetic network analysis of SARS-CoV-2 genomes In a phylogenetic network analysis of 160 complete human severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) genomes, we find three central variants distinguished by amino acid changes, which we have named A, B, and C, with A being the ancestral type according to the bat outgroup coronavirus. The A and C types are found in significant proportions outside East Asia, that is, in Europeans and Americans. In contrast, the B type is the most common type in East Asia, and its ancestral genome appears not to have spread outside East Asia without first mutating into derived B types, pointing to founder effects or immunological or environmental resistance against this type outside Asia. The network faithfully traces routes of infections for documented coronavirus disease 2019 (COVID-19) cases, indicating that phylogenetic networks can likewise be successfully used to help trace undocumented COVID-19 infection sources, which can then be quarantined to prevent recurrent spread of the disease worldwide.

positive: Chasing the origin of SARS-CoV-2 in Canada’s COVID-19 cases: A genomics study The emergence and global spread of SARS-CoV-2 has had profound social and economic consequences and has shed light on the importance of continued and additional investment in global health and infectious disease surveillance. Identifying changes in viral genomes provides key insights into viral diversity, how viruses spread within populations, and viral strategies for evasion of host immune systems. Here we report twenty-five SARS-CoV-2 genome sequences collected from some of the first COVID-19 cases in eastern Ontario, Canada (March 18-30, 2020). The reported genomes belong to the S-clade (n=2) and G-clade (n=23) of SARS-CoV-2 and contain 45 polymorphic sites including one shared missense and three unique synonymous variants in the gene encoding the spike protein. A phylogenetic analysis enabled the tracing of viral origin and potential transmission into and within Canada. There may be as many as sixteen unique infection events represented in these samples, including at least three that were likely introduced from Europe and seven from the USA. In addition, four separate genomes are each shared by multiple patients, suggesting a common origin or community spread even during this early stage of infection. These results demonstrate how molecular epidemiology and evolutionary phylogenetics can help local health units track origins and vectors of spread for emerging diseases like SARS-CoV-2. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to prevent future pandemics.

positive: Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology Since its emergence and detection in Wuhan, China in late 2019, the novel coronavirus SARS-CoV-2 has spread to nearly every country around the world, resulting in hundreds of thousands of infections to date. The virus was first detected in the Pacific Northwest region of the United States in January, 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the U.S., we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated impacts of federal travel restrictions. This study provides evidence for widespread, sustained transmission of SARS-CoV-2 within the U.S. and highlights the critical need for local surveillance.

positive: A snapshot of SARS-CoV-2 genome availability up to 30th March, 2020 and its implications The SARS-CoV-2 pandemic has been growing exponentially, affecting nearly 900 thousand people and causing enormous distress to economies and societies worldwide. A plethora of analyses based on viral sequences has already been published, in scientific journals as well as through non-peer reviewed channels, to investigate SARS-CoV-2 genetic heterogeneity and spatiotemporal dissemination. We examined full genome sequences currently available to assess the presence of sufficient information for reliable phylogenetic and phylogeographic studies in countries with the highest toll of confirmed cases. Although number of-available full-genomes is growing daily, and the full dataset contains sufficient phylogenetic information that would allow reliable inference of phylogenetic relationships, country-specific SARS-CoV-2 datasets still present severe limitations. Studies assessing within country spread or transmission clusters should be considered preliminary at best, or hypothesis generating. Hence the need for continuing concerted efforts to increase number and quality of the sequences required for robust tracing of the epidemic. Significance Statement Although genome sequences of SARS-CoV-2 are growing daily and contain sufficient phylogenetic information, country-specific data still present severe limitations and should be interpreted with caution.

positive: A simple method for detection of a novel coronavirus (SARS-CoV-2) using one-step RT-PCR followed by restriction fragment length polymorphism A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. Early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. We aimed to establish a simple method for the detection of SARS-CoV-2 in differentiating with SARS-CoV. Primers of our in-house reverse transcription polymerase chain reaction (RT-PCR) assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I, and AluI to distinguish between SARS-CoV-2 and SARS-CoV. In this report, a 396-bp fragment of the RdRp gene and 345-bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP-amplified product of SARS-CoV-2 generating three fragments of 45, 154, and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating two fragments of 248 and 97 bp without cutting to SARS-CoV. The accuracy of the assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, in the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV.

positive: About the origin of the first two Sars-CoV-2 infections in Italy: inference not supported by appropriate sequence analysis In the 5th February 2020 issue of Journal of Medical Virology a paper was published by Giovannetti et al., entitled "The first two cases of 2019-nCoV in Italy: where they come from?"1 . In this paper a phylogenetic and evolutionary analysis was applied to the virus identified in the first two subjects diagnosed in Italy with 2019-nCoV infection, recently renamed SARS-CoV-22 , two Chinese spouses arrived in Italy for tourism. The diagnosis was performed by the virology team under direction of Maria R. Capobianchi, at the National Institute of Infectious Diseases (INMI) in Rome, Italy, where the patients are currently hospitalized. This article is protected by copyright. All rights reserved.

positive: The novel Coronavirus enigma: Phylogeny and mutation analyses of SARS-CoV-2 viruses circulating in India during early 2020 Background This is a comprehensive analysis of 46 Indian SARS-CoV-2 genome sequences available from the NCBI and GISAID repository during early 2020. Evolutionary dynamics, gene-specific phylogeny and emergence of the novel co-evolving mutations in nine structural and non-structural genes among circulating SARS-CoV-2 strains in ten states of India have been assessed. Materials and methods 46 SARS-CoV-2 nucleotide sequences submitted from India were downloaded from the GISAID (39/46) or from NCBI (7/46) database. Phylogenetic study and analyses of mutation were based on the nine structural and non-structural genes of SARS-CoV-2 strains. Secondary structure of RdRP/NSP12 protein was predicted with respect to the novel A97V mutation. Results Phylogenetic analyses revealed the evolution of “genome-type clusters” and adaptive selection of “L” type SARS-CoV-2 strains with genetic closeness to the bat SARS-like coronaviruses than pangolin or MERS-CoVs. With regards to the novel co-evolving mutations, 2 groups are seen to circulate in India at present: the “major group” (52.2%) and the “minor group” (30.4%), harboring four and five co-existing mutations, respectively. The “major group” mutations fall in the A2a clade. All the minor group mutations, except 11083G>T (L37F, NSP6) were unique to the Indian isolates. Conclusion The study highlights rapidly evolving SARS-CoV-2 virus and co-circulation of multiple clades and sub-clades, driving this pandemic worldwide. This comprehensive study is a potential resource for monitoring the novel mutations in the viral genome, changes in viral pathogenesis, for designing vaccines and other therapeutics.

positive: Genome sequencing of the first SARS-CoV-2 reported from patients with COVID-19 in Ecuador SARS-CoV-2, the etiological agent of COVID-19 was first described in Wuhan in December 2019 and has now spread globally. Ecuador was the second country in South America to report confirmed cases. The first case reported in Quito, the capital city of Ecuador, was a tourist who came from the Netherlands and presented symptoms on March 10th, 2020 (index case). In this work we used the MinION platform (Oxford Nanopore Technologies) to sequence the metagenome of the bronchoalveolar lavage (BAL) from this case reported, and subsequently we sequenced the whole genome of the index case and other three patients using the ARTIC network protocols. Our data from the metagenomic approach confirmed the presence of SARS-CoV-2 coexisting with pathogenic bacteria suggesting coinfection. Relevant bacteria found in the BAL metagenome were Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus aureus and Chlamydia spp. Lineage assignment of the four whole genomes revealed three different origins. The variant HEE-01 was imported from the Netherlands and was assigned to B lineage, HGSQ-USFQ-018, belongs to the B.1 lineage showing nine nucleotide differences with the reference strain and grouped with sequences from the United Kingdom, and HGSQ-USFQ-007 and HGSQ-USFQ-010 belong to the B lineage and grouped with sequences from Scotland. All genomes show mutations in their genomes compared to the reference strain, which could be important to understand the virulence, severity and transmissibility of the virus. Our findings also suggest that there were at least three independent introductions of SARS-CoV-2 to Ecuador.

positive: Transmission dynamics and evolutionary history of 2019-nCoV To investigate the time origin, genetic diversity, and transmission dynamics of the recent 2019-nCoV outbreak in China and beyond, a total of 32 genomes of virus strains sampled from China, Thailand, and the USA with sampling dates between 24 December 2019 and 23 January 2020 were analyzed. Phylogenetic, transmission network, and likelihood-mapping analyses of the genome sequences were performed. On the basis of the likelihood-mapping analysis, the increasing tree-like signals (from 0% to 8.2%, 18.2%, and 25.4%) over time may be indicative of increasing genetic diversity of 2019-nCoV in human hosts. We identified three phylogenetic clusters using the Bayesian inference framework and three transmission clusters using transmission network analysis, with only one cluster identified by both methods using the above genome sequences of 2019-nCoV strains. The estimated mean evolutionary rate for 2019-nCoV ranged from 1.7926 × 10-3 to 1.8266 × 10-3 substitutions per site per year. On the basis of our study, undertaking epidemiological investigations and genomic data surveillance could positively impact public health in terms of guiding prevention efforts to reduce 2019-nCOV transmission in real-time.

positive: Multivariate Analyses of Codon Usage of SARS-CoV-2 and other betacoronaviruses Coronavirus disease 2019 (COVID-19) is a global health concern as it continues to spread within China and beyond. The causative agent of this disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus which also includes severe acute respiratory syndrome related coronavirus (SARSr-CoV) and Middle East respiratory syndrome related coronavirus (MERSr-CoV). Codon usage of viral genes are believed to be subjected to different selection pressures in different host environments. Previous studies on codon usage of influenza A viruses can help identify viral host origins and evolution trends, however, similar studies on coronaviruses are lacking. In this study, global correspondence analysis (CA), within-group correspondence analysis (WCA) and between-group correspondence analysis (BCA) were performed among different genes in coronavirus viral sequences. The amino acid usage pattern of SARS-CoV-2 was generally found similar to bat and human SARSr-CoVs. However, we found greater synonymous codon usage differences between SARS-CoV-2 and its phylogenetic relatives on spike and membrane genes, suggesting these two genes of SARS-CoV-2 are subjected to different evolutionary pressures.

positive: Opinion paper: Severe Acute Respiratory Syndrome Coronavirus 2 and domestic animals: what relation?

positive: COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.

positive: The genetic sequence, origin, and diagnosis of SARS-CoV-2 Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a new infectious disease that first emerged in Hubei province, China, in December 2019, which was found to be associated with a large seafood and animal market in Wuhan. Airway epithelial cells from infected patients were used to isolate a novel coronavirus, named the SARS-CoV-2, on January 12, 2020, which is the seventh member of the coronavirus family to infect humans. Phylogenetic analysis of full-length genome sequences obtained from infected patients showed that SARS-CoV-2 is similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and uses the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), as SARS-CoV. The possible person-to-person disease rapidly spread to many provinces in China as well as other countries. Without a therapeutic vaccine or specific antiviral drugs, early detection and isolation become essential against novel Coronavirus. In this review, we introduced current diagnostic methods and criteria for the SARS-CoV-2 in China and discuss the advantages and limitations of the current diagnostic methods, including chest imaging and laboratory detection.

positive: Phylogenetic clustering of the Indian SARS-CoV-2 genomes reveals the presence of distinct clades of viral haplotypes among states The first Indian cases of COVID-19 caused by SARS-Cov-2 were reported in February 29, 2020 with a history of travel from Wuhan, China and so far above 4500 deaths have been attributed to this pandemic. The objectives of this study were to characterize Indian SARS-CoV-2 genome-wide nucleotide variations, trace ancestries using phylogenetic networks and correlate state-wise distribution of viral haplotypes with differences in mortality rates. A total of 305 whole genome sequences from 19 Indian states were downloaded from GISAID. Sequences were aligned using the ancestral Wuhan-Hu genome sequence (NC_045512.2). A total of 633 variants resulting in 388 amino acid substitutions were identified. Allele frequency spectrum, and nucleotide diversity (π) values revealed the presence of higher proportions of low frequency variants and negative Tajima’s D values across ORFs indicated the presence of population expansion. Network analysis highlighted the presence of two major clusters of viral haplotypes, namely, clade G with the S:D614G, RdRp: P323L variants and a variant of clade L [Lv] having the RdRp:A97V variant. Clade G genomes were found to be evolving more rapidly into multiple sub-clusters including clade GH and GR and were also found in higher proportions in three states with highest mortality rates namely, Gujarat, Madhya Pradesh and West Bengal.

positive: Genomic variations of SARS-CoV-2 suggest multiple outbreak sources of transmission We examined 169 genomes of SARS-CoV-2 and found that they can be classified into two major genotypes, Type I and Type II. Type I can be further divided into Type IA and IB. Our phylogenetic analysis showed that the Type IA resembles the ancestral SARS-CoV-2 most. Type II was likely evolved from Type I and predominant in the infections. Our results suggest that Type II SARS-CoV-2 was the source of the outbreak in the Wuhan Huanan market and it was likely originated from a super-spreader. The outbreak caused by the Type I virus should have occurred somewhere else, because the patients had no direct link to the market. Furthermore, by analyzing three genomic sites that distinguish Type I and Type II strains, we found that synonymous changes at two of the three sites confer higher protein translational efficiencies in Type II strains than in Type I strains, which might explain why Type II strains are predominant, implying that Type II is more contagious (transmissible) than Type I. These findings could be valuable for the current epidemic prevention and control.

positive: Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective SARS-CoV-2, a new coronavirus strain responsible for COVID-19 has emerged in Wuhan City, China and still continuing its worldwide pandemic nature. Considering the severity of the disease, a number of studies are underway, and full genomic sequences have already been released in the last few weeks to enable the understanding of the evolutionary origin and molecular characteristics of this virus. Bioinformatics analysis, satellite derived imaging data and epidemiological attributes were employed to investigate origin, immunogenic resemblance and global threat of newly pandemic SARS-CoV-2 including Bangladesh perspective. Based on currently available genomic information, a phylogeny study was employed focusing four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings clearly demonstrated that SARS-CoV-2 exhibited evolutionary convergent relation with previously reported SARS-CoV. It was also found that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed lower level of similarity and identical patterns. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. The study also prioritized the temperature comparison through satellite imaging alongside compiling and analyzing the epidemiological outbreak information on the 2019 novel coronavirus based on several open datasets on COVID-19 (SARS-CoV-2) and discussed possible threats to Bangladesh.

positive: No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2 The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2, which jumped into the human population in late 2019 from a currently uncharacterised reservoir. Due to this extremely recent association with humans, SARS-CoV-2 may not yet be fully adapted to its human host. This has led to speculations that some lineages of SARS-CoV-2 may be evolving towards higher transmissibility. The most plausible candidate mutations under putative natural selection are those which have emerged repeatedly and independently (homoplasies). Here, we formally test whether any of the recurrent mutations that have been observed in SARS-CoV-2 to date are significantly associated with increased viral transmission. To do so, we developed a phylogenetic index to quantify the relative number of descendants in sister clades with and without a specific allele. We apply this index to a carefully curated set of recurrent mutations identified within a dataset of over 23,000 SARS-CoV-2 genomes isolated from patients worldwide. We do not identify a single recurrent mutation in this set convincingly associated with increased viral transmission. Instead, recurrent SARS-CoV-2 mutations currently in circulation appear to be evolutionary neutral. Recurrent mutations also seem primarily induced by the human immune system via host RNA editing, rather than being signatures of adaption to the novel human host. We find no evidence at this stage for the emergence of more transmissible lineages of SARS-CoV-2 due to recurrent mutations.

positive: The origin and early spread of SARS-CoV-2 in Europe The investigation of migratory patterns of the SARS-CoV-2 pandemic before border closures in Europe is a crucial first step towards an in-depth evaluation of border closure policies. Here we analyze viral genome sequences using a phylodynamic model with geographic structure to estimate the origin and spread of SARS-CoV-2 in Europe prior to border closures. Based on SARS-CoV-2 genomes, we reconstruct a partial transmission tree of the early pandemic, including inferences of the geographic location of ancestral lineages and the number of migration events into and between European regions. We find that the predominant lineage spreading in Europe has a most recent common ancestor in Italy and was probably seeded by a transmission event in either Hubei or Germany. We do not find evidence for preferential migration paths from Hubei into different European regions or from each European region to the others. Sustained local transmission is first evident in Italy and then shortly thereafter in the other European regions considered. Before the first border closures in Europe, we estimate that the rate of occurrence of new cases from within-country transmission was within the bounds of the estimated rate of new cases from migration. In summary, our analysis offers a view on the early state of the epidemic in Europe and on migration patterns of the virus before border closures. This information will enable further study of the necessity and timeliness of border closures.

positive: Phyloevolutionary analysis of SARS-CoV-2 in Nigeria Abstract Background Phyloepidemiologic approaches have given specific insight to understanding emergence and evolution of infection. Knowledge on the outbreak and spread of SARS-CoV-2 in Nigeria would assist in providing preventive measures to reduce transmission among populations at risk. Therefore, this study aimed at investigating the evolution of SARS-CoV-2 in Nigeria. Materials and Method A total of 39 complete genomes of SARS-CoV-2 were retrieved from the GISAID EpiFluTM database on March 29th 2020 to investigate its evolution in Nigeria. Sequences were selected based on the travel history of the patient and the collection date. Other sequences were not selected because they were short, contained artefacts, not from original source or had insufficient information. Evolutionary history was inferred using Maximum Likelihood method based on the General Time Reversible model. Phylogenetic tree was constructed to determine the common ancestor of each strain. Results The phylogenetic analysis showed the strain in Nigeria clustered in a monophyletic clade with a Wuhan sublineage. Nucleotide alignment also showed a 100% similarity indicating a common origin of evolution. Comparative analysis showed 27,972 (93.6%) identical sites and 97.6% pairwise identity with the consensus. Conclusion The study evidently showed the entire outbreak of COVID-19 infection in Nigeria stemmed from a single introduction sharing consensus similarity with the reference SARS-CoV-2 human genome from Wuhan. Preventive measures that can limit the spread of the infection among populations at risk should be implemented.

positive: Genetic evolution analysis of 2019 novel coronavirus and coronavirus from other species The Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a Public Health Emergency of International Concern. However, so far, there are still controversies about the source of the virus and its intermediate host. Here, we found the novel coronavirus was closely related to coronaviruses derived from five wild animals, including Paguma larvata, Paradoxurus hermaphroditus, Civet, Aselliscus stoliczkanus and Rhinolophus sinicus, and was in the same branch of the phylogenetic tree. However, genome and ORF1a homology show that the virus is not the same coronavirus as the coronavirus derived from these five animals, whereas the virus has the highest homology with Bat coronavirus isolate RaTG13.

positive: Transmission dynamics and evolutionary history of 2019‐nCoV To investigate the time origin, genetic diversity, and transmission dynamics of the recent 2019‐nCoV outbreak in China and beyond, a total of 32 genomes of virus strains sampled from China, Thailand, and the USA with sampling dates between 24 December 2019 and 23 January 2020 were analyzed. Phylogenetic, transmission network, and likelihood‐mapping analyses of the genome sequences were performed. On the basis of the likelihood‐mapping analysis, the increasing tree‐like signals (from 0% to 8.2%, 18.2%, and 25.4%) over time may be indicative of increasing genetic diversity of 2019‐nCoV in human hosts. We identified three phylogenetic clusters using the Bayesian inference framework and three transmission clusters using transmission network analysis, with only one cluster identified by both methods using the above genome sequences of 2019‐nCoV strains. The estimated mean evolutionary rate for 2019‐nCoV ranged from 1.7926 × 10(−3) to 1.8266 × 10(−3) substitutions per site per year. On the basis of our study, undertaking epidemiological investigations and genomic data surveillance could positively impact public health in terms of guiding prevention efforts to reduce 2019‐nCOV transmission in real‐time.

positive: SARS‐CoV‐2 spike protein favors ACE2 from Bovidae and Cricetidae Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes the recent COVID‐19 public health crisis. Bat is the widely believed original host of SARS‐CoV‐2. However, its intermediate host before transmitting to humans is not clear. Some studies proposed pangolin, snake, or turtle as the intermediate hosts. Angiotensin‐converting enzyme 2 (ACE2) is the receptor for SARS‐CoV‐2, which determines the potential host range for SARS‐CoV‐2. On the basis of structural information of the complex of human ACE2 and SARS‐CoV‐2 receptor‐binding domain (RBD), we analyzed the affinity to S protein of the 20 key residues in ACE2 from mammal, bird, turtle, and snake. Several ACE2 proteins from Primates, Bovidae, Cricetidae, and Cetacea maintained the majority of key residues in ACE2 for associating with SARS‐CoV‐2 RBD. The simulated structures indicated that ACE2 proteins from Bovidae and Cricetidae were able to associate with SARS‐CoV‐2 RBD. We found that nearly half of the key residues in turtle, snake, and bird were changed. The simulated structures showed several key contacts with SARS‐CoV‐2 RBD in turtle and snake ACE2 were abolished. This study demonstrated that neither snake nor turtle was the intermediate hosts for SARS‐CoV‐2, which further reinforced the concept that the reptiles are resistant against infection of coronavirus. This study suggested that Bovidae and Cricetidae should be included in the screening of intermediate hosts for SARS‐CoV‐2.

positive: A study on the relationship between BCG vaccination and Covid-19 prevalence: Do other confounders warrant investigation? The Covid-19 pandemic, which originated from Wuhan, Hubei province, China, and quickly spread to the rest of the globe is caused by SARS-CoV-2, a single-stranded RNA virus. Preliminary data suggest a relationship between the BCG vaccine and the prevalence of Covid-19. The BCG vaccine is used in the prevention of tuberculosis, a disease that is most prevalent in developing countries. To determine the potential protective role of BCG vaccination, this study investigated the occurrence of Covid-19 and the relationship between the spread of Covid-19 in countries that offer BCG vaccination and those that do not. The study also performed a phylogenetic analysis of the strains involved in the Covid-19 outbreak from the representative countries. To achieve the objectives, the study utilized publicly available data on population size, vaccination coverage, and Covid-19 cases. Phylogenetic analysis was used to determine if some SARS-CoV-2 strains were more prevalent than others. The study revealed a significant negative trend between countries that offer the BCG vaccine to the general population and the reported cases of Covid-19. The study proposes future molecular and immunological analyses to determine the potential role of BCG vaccination in protection against Covid-19. This will determine if BCG has antiviral properties, with the possibility of recommending it for widespread use if supported by scientific data.

positive: Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design Novel coronavirus has wrecked medical and health care facilities claiming â¼5% death tollsglobally. All efforts to contain the pathogenesis either using inhibitory drugs or vaccines largelyremained futile due to a lack of better understanding of the genomic feature of this virus. In thepresent study, we compared the 2019-nCoV with other coronaviruses, which indicated that batSARS like coronavirus could be a probable ancestor of the novel coronavirus. The proteinsequence similarity of pangolin-hCoV and bat-hCoV with human coronavirus was higher ascompared to their nucleotide similarity denoting the occurrence of more synonymous mutationsin the genome. Phylogenetic and alignment analysis of 591 novel coronaviruses of differentclades from Group I to Group V revealed several mutations and concomitant amino acidchanges. Detailed investigation on nucleotide substitution unfolded 100 substitutions in thecoding region of which 43 were synonymous and 57 were of non-synonymous type. The nonsynonymous substitutions resulting into 57 amino acid changes were found to be distributed overdifferent hCoV proteins with maximum on spike protein. An important di-amino acid change RGto KR was observed in ORF9 protein. Additionally, several interesting features of the novelcoronavirus genome have been highlighted in respect to various other human infecting viruseswhich may explain extreme pathogenicity, infectivity and simultaneously the reason behindfailure of the antiviral therapies. SUMMARY: This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. The analysis unravel various amino acid mutations in the viral proteins which may provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.

positive: Pangolin homology associated with 2019-nCoV To explore potential intermediate host of a novel coronavirus is vital to rapidly control continuous COVID-19 spread. We found genomic and evolutionary evidences of the occurrence of 2019-nCoV-like coronavirus (named as Pangolin-CoV) from dead Malayan Pangolins. Pangolin-CoV is 91.02% and 90.55% identical at the whole genome level to 2019-nCoV and BatCoV RaTG13, respectively. Pangolin-CoV is the lowest common ancestor of 2019-nCoV and RaTG13. The S1 protein of Pangolin-CoV is much more closely related to 2019-nCoV than RaTG13. Five key amino-acid residues involved in the interaction with human ACE2 are completely consistent between Pangolin-CoV and 2019-nCoV but four amino-acid mutations occur in RaTG13. It indicates Pangolin-CoV has similar pathogenic potential to 2019-nCoV, and would be helpful to trace the origin and probable intermediate host of 2019-nCoV.

positive: Bioinformatic analysis indicates that SARS-CoV-2 is unrelated to known artificial coronaviruses OBJECTIVE: SARS-CoV-2 is responsible for the present coronavirus pandemic and some suggestions were made about its possible artificial origin. We, therefore, compared SARS-CoV-2 with such known viruses that were prepared in the laboratory and other relevant natural strains to estimate their genetic relatedness. MATERIALS AND METHODS: BLAST and clustalW were used to identify and align viral sequences of SARS-CoV-2 to other animal coronaviruses (human, bat, mouse, pangolin) and related artificial constructs. Phylogenetics trees were then prepared using iTOL. RESULTS: Our study supports the notion that known artificial coronaviruses, including the chimeric SL-SHC014-MA15 synthesized in 2015, differ too much from SARS-CoV-2 to hypothesize an artificial origin of the latter. On the contrary, our data support the natural origin of the COVID-19 virus, likely derived from bats, possibly transferred to pangolins, before spreading to man. CONCLUSIONS: Speculations about the artificial origin of SARS-CoV-2 are most likely unfounded. On the contrary, when carefully handled, engineered organisms provide a unique opportunity to study biological systems in a controlled fashion. Biotechnology is a powerful tool to advance medical research and should not be abandoned because of irrational fears.

positive: Stability of SARS-CoV-2 Phylogenies The SARS-CoV-2 pandemic has led to unprecedented, nearly real-time genetic tracing due to the rapid community sequencing response. Researchers immediately leveraged these data to infer the evolutionary relationships among viral samples and to study key biological questions, including whether host viral genome editing and recombination are features of SARS-CoV-2 evolution. This global sequencing effort is inherently decentralized and must rely on data collected by many labs using a wide variety of molecular and bioinformatic techniques. There is thus a strong possibility that systematic errors associated with lab-specific practices affect some sequences in the repositories. We find that some recurrent mutations in reported SARS-CoV-2 genome sequences have been observed predominantly or exclusively by single labs, co-localize with commonly used primer binding sites and are more likely to affect the protein coding sequences than other similarly recurrent mutations. We show that their inclusion can affect phylogenetic inference on scales relevant to local lineage tracing, and make it appear as though there has been an excess of recurrent mutation and/or recombination among viral lineages. We suggest how samples can be screened and problematic mutations removed. We also develop tools for comparing and visualizing differences among phylogenies and we show that consistent clade- and tree-based comparisons can be made between phylogenies produced by different groups. These will facilitate evolutionary inferences and comparisons among phylogenies produced for a wide array of purposes. Building on the SARS-CoV-2 Genome Browser at UCSC, we present a toolkit to compare, analyze and combine SARS-CoV-2 phylogenies, find and remove potential sequencing errors and establish a widely shared, stable clade structure for a more accurate scientific inference and discourse. Foreword We wish to thank all groups that responded rapidly by producing these invaluable and essential sequence data. Their contributions have enabled an unprecedented, lightning-fast process of scientific discovery---truly an incredible benefit for humanity and for the scientific community. We emphasize that most lab groups with whom we associate specific suspicious alleles are also those who have produced the most sequence data at a time when it was urgently needed. We commend their efforts. We have already contacted each group and many have updated their sequences. Our goal with this work is not to highlight potential errors, but to understand the impacts of these and other kinds of highly recurrent mutations so as to identify commonalities among the suspicious examples that can improve sequence quality and analysis going forward.

positive: Phylogenetic analysis of the full‐length SARS‐CoV sequences: Evidence for phylogenetic discordance in three genomic regions The origin of the severe acute respiratory syndrome‐coronavirus (SARS‐CoV) remains unclear. Evidence based on Bayesian scanning plots and phylogenetic analysis using maximum likelihood (ML) and Bayesian methods indicates that SARS‐CoV, for the largest part of the genome (∼80%), is more closely related to Group II coronaviruses sequences, whereas in three regions in the ORF1ab gene it shows no apparent similarity to any of the previously characterized groups of coronaviruses. There is discordant phylogenetic clustering of SARS‐CoV and coronaviruses sequences, throughout the genome, compatible with either ancient recombination events or altered evolutionary rates in different lineages, or a combination of both. J. Med. Virol. 74:369–372, 2004. © 2004 Wiley‐Liss, Inc.

positive: How related is SARS-CoV-2 to other coronaviruses?

positive: Early phylogenetic estimate of the effective reproduction number of SARS-CoV-2 To reconstruct the evolutionary dynamics of the 2019 novel-coronavirus recently causing an outbreak in Wuhan, China, 52 SARS-CoV-2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent-based exponential growth and a birth-death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10-4 subs/site/year (range, 1.1 × 10-4 -15 × 10-4 ) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1-5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.

positive: SARS‐CoV‐2: Structural diversity, phylogeny, and potential animal host identification of spike glycoprotein To investigate the evolutionary history of the current pandemic outbreak of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a total of 137 genomes of coronavirus strains with release dates between January 2019 and 25 March 2020, were analyzed. To investigate the potential intermediate host of the SARS‐CoV‐2, we analyzed spike glycoprotein sequences from different animals, with particular emphasis on bats. We performed phylogenetic analysis and structural reconstruction of the spike glycoproteins with subsequent alignment and comparison. Our phylogenetic results revealed that SARS‐CoV‐2 was more similar to the bats' betacoronavirus isolates: HKU5‐related from Pipistrellus abramus and HKU4‐related from Tylonycteris pachypus. We also identified a yak betacoronavirus strain, YAK/HY24/CH/2017, as the closest match in the comparison of the structural models of spike glycoproteins. Interestingly, a set of unique features has been described for this particular strain of the yak betacoronavirus. Therefore, our results suggest that the human SARS‐CoV‐2, responsible for the current outbreak of COVID‐19, could also come from yak as an intermediate host.

positive: SARS-CoV-2 genomic surveillance in Taiwan revealed novel ORF8-deletion mutant and clade possibly associated with infections in Middle East Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

positive: Structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-nCoV) and SARS coronavirus (SARS-CoV) The emergence of 2019 novel coronavirus (2019-nCoV) is of global concern and might have emerged from RNA recombination among existing coronaviruses. CoV spike (S) protein which is crucial for receptor binding, membrane fusion via conformational changes, internalization of the virus, host tissue tropism and comprises crucial targets for vaccine development, remain largely uncharacterized. Therefore, the present study has been planned to determine the sequence variation, structural and antigenic divergence of S glycoprotein which may be helpful for the management of 2019-nCoV infection. The sequences of spike glycoprotein of 2019-nCoV and SARS coronavirus (SARS-CoV) were used for the comparison. The sequence variations were determined using EMBOSS Needle pairwise sequence alignment tools. The variation in glycosylation sites was predicted by NetNGlyc 1.0 and validated by N-GlyDE server. Antigenicity was predicted by NetCTL 1.2 and validated by IEDB Analysis Resource server. The structural divergence was determined by using SuperPose Version 1.0 based on cryo-EM structure of the SARS coronavirus spike glycoprotein. Our data suggests that 2019-nCoV is newly spilled coronavirus into humans in China is closely related to SARS-CoV, which has only 12.8% of difference with SARS-CoV in S protein and has 83.9% similarity in minimal receptor-binding domain with SARS-CoV. Addition of a novel glycosylation sites were observed in 2019-nCoV. In addition, antigenic analysis proposes that great antigenic differences exist between both the viral strains, but some of the epitopes were found to be similar between both the S proteins. In spite of the variation in S protein amino acid composition, we found no significant difference in their structures. Collectively, for the first time our results exhibit the emergence of human 2019-nCoV is closely related to predecessor SARS-CoV and provide the evidence that 2019-nCoV uses various novel glycosylation sites as SARS-CoV and may have a potential to become pandemic owing its antigenic discrepancy. Further, demonstration of novel Cytotoxic T lymphocyte epitopes may impart opportunities for the development of peptide based vaccine for the prevention of 2019-nCoV.

positive: Network-based Drug Repurposing for Human Coronavirus Human Coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle east respiratory syndrome coronavirus (MERS-CoV), and 2019 novel coronavirus (2019-nCoV), lead global epidemics with high morbidity and mortality. However, there are currently no effective drugs targeting 2019-nCoV. Drug repurposing, represented as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. In this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the HCoV-host interactome and drug targets in the human protein-protein interaction network. Phylogenetic analyses of 15 HCoV whole genomes reveal that 2019-nCoV has the highest nucleotide sequence identity with SARS-CoV (79.7%) among the six other known pathogenic HCoVs. Specifically, the envelope and nucleocapsid proteins of 2019-nCoV are two evolutionarily conserved regions, having the sequence identities of 96% and 89.6%, respectively, compared to SARS-CoV. Using network proximity analyses of drug targets and known HCoV-host interactions in the human protein-protein interactome, we computationally identified 135 putative repurposable drugs for the potential prevention and treatment of HCoVs. In addition, we prioritized 16 potential anti-HCoV repurposable drugs (including melatonin, mercaptopurine, and sirolimus) that were further validated by enrichment analyses of drug-gene signatures and HCoV-induced transcriptomics data in human cell lines. Finally, we showcased three potential drug combinations (including sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the Complementary Exposure pattern: the targets of the drugs both hit the HCoV-host subnetwork, but target separate neighborhoods in the human protein-protein interactome network. In summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations toward future clinical trials for HCoVs.

positive: Analysis of RNA sequences of 3636 SARS-CoV-2 collected from 55 countries reveals selective sweep of one virus type. Background & objectives SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) is evolving with the progression of the pandemic. This study was aimed to investigate the diversity and evolution of the coronavirus SARS-CoV-2 with progression of the pandemic over time and to identify similarities and differences of viral diversity and evolution across geographical regions (countries). Methods Publicly available data on type definitions based on whole-genome sequences of the SARS-CoV-2 sampled during December and March 2020 from 3636 infected patients spread over 55 countries were collected. Phylodynamic analyses were performed and the temporal and spatial evolution of the virus was examined. Results It was found that (i) temporal variation in frequencies of types of the coronavirus was significant; ancestral viruses of type O were replaced by evolved viruses belonging to type A2a; (ii) spatial variation was not significant; with the spread of SARS-CoV-2, the dominant virus was the A2a type virus in every geographical region; (iii) within a geographical region, there was significant micro-level variation in the frequencies of the different viral types, and (iv) the evolved coronavirus of type A2a swept rapidly across all continents. Interpretation & conclusions SARS-CoV-2 belonging to the A2a type possesses a non-synomymous variant (D614G) that possibly eases the entry of the virus into the lung cells of the host. This may be the reason why the A2a type has an advantage to infect and survive and as a result has rapidly swept all geographical regions. Therefore, large-scale sequencing of coronavirus genomes and, as required, of host genomes should be undertaken in India to identify regional and ethnic variation in viral composition and its interaction with host genomes. Further, careful collection of clinical and immunological data of the host can provide deep learning in relation to infection and transmission of the types of coronavirus genomes.

positive: Coronavirus3D: 3D structural visualization of COVID-19 genomic divergence MOTIVATION: As the COVID-19 pandemics is spreading around the world, the SARS-CoV-2 virus is evolving with mutations that potentially change and fine-tune functions of the proteins coded in its genome. RESULTS: Coronavirus3D website integrates data on the SARS-CoV-2 virus mutations with information about 3D structures of its proteins, allowing users to visually analyze the mutations in their 3D context. AVAILABILITY: Coronavirus3D server is freely available at https://coronavirus3d.org.

positive: A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted.

positive: Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States Summary The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

positive: Genome analysis of SARS-CoV-2 isolates occurring in India: Present scenario. Background The origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still a debatable topic. The association of the virus spread from the market is supported by the close relation of genome sequences of environmental surface samples with virus samples from earliest patients by phylogenetic analysis. Objectives To have an insight into the SARS-CoV-2 genome sequences reported from India for better understanding on their epidemiology and virulence. Methods Genome sequences of Indian isolates of SARS-CoV-2 were analyzed to understand their phylogeny and divergence with respect to other isolates reported from other countries. Amino acid sequences of individual open reading frames (ORFs) from SARS-CoV-2 Indian isolates were aligned with sequences of isolates reported from other countries to identify the mutations occurred in Indian isolates. Results Our analysis suggests that Indian SARS-CoV-2 isolates are closely related to isolates reported from other parts of the world. Most ORFs are highly conserved; mutations were also detected in some ORFs. We found that most isolates reported from India have key mutations at 614th position of the S protein and 84th position of the ORF 8, which has been reported to be associated with high virulence and high transmission rate. Conclusion An attempt was made to understand the SARS-CoV-2 virus reported from India. SARS-CoV-2 reported from India was closely similar to other SARS-CoV-2 reported from other parts of the world, which suggests that vaccines and other therapeutic methods generated from other countries might work well in India. In addition, available sequence data suggest that majority of Indian isolates are capable of high transmission and virulence.

positive: An exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human‐pathogenic coronavirus (SARS‐CoV‐2) The city of Wuhan, Hubei province, China, was the origin of a severe pneumonia outbreak in December 2019, attributed to a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]), causing a total of 2761 deaths and 81109 cases (25 February 2020). SARS‐CoV‐2 belongs to genus Betacoronavirus, subgenus Sarbecovirus. The polyprotein 1ab (pp1ab) remains unstudied thoroughly since it is similar to other sarbecoviruses. In this short communication, we performed phylogenetic‐structural sequence analysis of pp1ab protein of SARS‐CoV‐2. The analysis showed that the viral pp1ab has not changed in most isolates throughout the outbreak time, but interestingly a deletion of 8 aa in the virulence factor nonstructural protein 1 was found in a virus isolated from a Japanese patient that did not display critical symptoms. While comparing pp1ab protein with other betacoronaviruses, we found a 42 amino acid signature that is only present in SARS‐CoV‐2 (AS‐SCoV2). Members from clade 2 of sarbecoviruses have traces of this signature. The AS‐SCoV2 located in the acidic‐domain of papain‐like protein of SARS‐CoV‐2 and bat‐SL‐CoV‐RatG13 guided us to suggest that the novel 2019 coronavirus probably emerged by genetic drift from bat‐SL‐CoV‐RaTG13. The implication of this amino acid signature in papain‐like protein structure arrangement and function is something worth to be explored.

positive: Genome-wide variations of SARS-CoV-2 infer evolution relationship and transmission route In the epidemic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the issues of mutation, origin, typing and the effect of mutation on molecular detection remain to be unrevealed. In order to identify the evolutionary relationship of SARS-CoV-2 and evaluate the detection efficiency of primers that are currently used in different countries, we retrieved genomic sequences of 373 SARS-CoV-2 strains from multiple databases and performed genome-wide variation analysis. According to the nucleotide C28144T variation, the SARS-CoV-2 can be divided into group A (117 strains) and group B (256 strains). The spike protein gene (S gene) coding region 1841 (total 23403) A1841G, formed a B1 subgroup (40 strains) in group B, of which 30 strains were from European and American countries in March (especially Washington, USA). These mutations are likely to be influenced by the environment or the immunization selection pressure of different populations. Although the mutation is not in the receptor binding region (RBD) and alkaline cleavage region, it may also affect the ability of transmission and pathogenicity; however, the significance is not yet clear. As the ratio of A / B strains in the epidemic months showed an increasing trend (0.35: 1 in January, 0.62: 1 in February and 0.76: 1 in March), it seems that the transmissibility of group A strains becomes stronger with time. Based on the variation of 11 nucleotide sites during the epidemic process, it is speculated that the Washington strain is more like an ancestor type, and the Wuhan strain is the offspring of the group A virus strain. By comparing the detection capabilities of primers in different countries, the SARS-CoV-2 nucleotide variation may only affect molecular detection of very few strains. The differences in the transmissibility, pathogenicity and clinical manifestations of different types of strains require further investigations.

positive: In-silico nucleotide and protein analyses of S-gene region in selected zoonotic coronaviruses reveal conserved domains and evolutionary emergence with trajectory course of viral entry from SARS-CoV2 genomic data The recent zoonotic coronavirus virus outbreak of a novel type [COVID 19] has necessitated the adequate understanding of the evolutionary pathway of zoonotic viruses which adversely affects human populations for therapeutic constructs to combat the pandemic now and in the future. We analyzed conserved domains of the severe acute respiratory coronavirus 2 [SARS-CoV2] for possible targets of viral entry inhibition in host cells, evolutionary relationship of human coronavirus [229E] and zoonotic coronaviruses with SAR-CoV2 as well as evolutionary relationship between selected SARS-CoV 2 genomic data. Conserved domains with antagonistic action on host innate antiviral cellular mechanisms in SARS-CoV 2 include nsp 11, nsp 13 etc. Also, multiple sequence alignments of the spike [S] gene protein of selected candidate zoonotic coronaviruses alongside the S gene protein of the SARs-CoV2 revealed closest evolutionary relationship [95.6%] with pangolin coronaviruses [S] gene. Clades formed between Wuhan SARS-CoV2 phylogeny data and five others suggests viral entry trajectory while revealing genomic and protein SARS CoV 2 data from Philippines as early ancestors. Therefore, phylogeny of SARS-CoV 2 genomic data suggests profiling in diverse populations with and without the outbreak alongside migration history and racial background for mutation tracking and dating of viral subtype divergence which is essential for effective management of present and future zoonotic coronavirus outbreaks.

positive: Phylogenetic analyses of the severe acute respiratory syndrome coronavirus 2 reflected the several routes of introduction to Taiwan, the United States, and Japan Worldwide Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is disrupting in the economy and anxiety of people. The public anxiety has increased the psychological burden on government and healthcare professionals, resulting in a government worker suicide in Japan. The terrified people are asking the government for border measures. However, are border measures possible for this virus? By analyzing 48 almost complete virus genome sequences, we found out that the viruses that invaded Taiwan, the United States, and Japan were introduced independently. We identified thirteen parsimony-informative sites and three groups (CTC, TCC, and TCT). Viruses found outside China did not form a monophyletic clade, opposite to previous study. These results suggest the difficulty of implementing effective border measures against this virus.

positive: Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission

positive: Emergence of multiple variants of SARS-CoV-2 with signature structural changes This study explores the divergence pattern of SARS-CoV-2 using whole genome sequences of the isolates from various COVID-19 affected countries. The phylogenomic analysis indicates the presence of at least four distinct groups of the SARS-CoV-2 genomes. The emergent groups have been found to be associated with signature structural changes in specific proteins. Also, this study reveals the differential levels of divergence patterns for the protein coding regions. Moreover, we have predicted the impact of structural changes on a couple of important viral proteins via structural modelling techniques. This study further advocates for more viral genetic studies with associated clinical outcomes and hosts’ response for better understanding of SARS-CoV-2 pathogenesis enabling better mitigation of this pandemic situation.

positive: Genomic epidemiology of SARS-CoV-2 spread in Scotland highlights the role of European travel in COVID-19 emergence Abstract SARS-CoV-2, the causative agent of COVID-19, emerged in Wuhan, China in December 2019 and spread rapidly throughout the world. Understanding the introductions of this new coronavirus in different settings may assist control efforts and the establishment of frameworks to support rapid response in future infectious disease outbreaks. We investigated the first four weeks of emergence of the SARS-CoV-2 virus in Scotland after the first case reported on the 1st March 2020. We obtained full genome sequences from 452 individuals with a laboratory-confirmed diagnosis of COVID-19, representing 20% of all cases until 1st April 2020 (n=2310). This permitted a genomic epidemiology approach to study the introductions and spread of the SARS-2 virus in Scotland. From combined phylogenetic and epidemiological analysis, we estimated at least 113 introductions of SARS-CoV-2 into Scotland during this period. Clusters containing multiple sequences suggestive of onward transmission occurred in 48/86 (56%). 42/86 (51%) clusters had no known international travel history indicating undetected introductions. The majority of viral sequences were most closely related to those circulating in other European countries, including Italy, Austria and Spain. Travel-associated introductions of SARS-CoV-2 into Scotland predated travel restrictions in the UK and other European countries. The first local transmission occurred three days after the first case. A shift from travel-associated to sustained community transmission was apparent after only 11 days. Undetected introductions occurred prior to the first known case of COVID-19. Earlier travel restrictions and quarantine measures might have resulted in fewer introductions into Scotland, thereby reducing the number of cases and the subsequent burden on health services. The high number of introductions and transmission rates were likely to have impacted on national contact tracing efforts. Our results also demonstrate that local real-time genomic epidemiology can be used to monitor transmission clusters and facilitate control efforts to restrict the spread of COVID-19.

positive: Emerging genetic diversity among clinical isolates of SARS-CoV-2: Lessons for today Considering the current pandemic of COVID-19, it is imperative to gauge the role of molecular divergence in SARS-CoV-2 with time, due to clinical and epidemiological concerns. Our analyses involving molecular phylogenetics is a step toward understanding the transmission clusters that can be correlated to pathophysiology of the disease to gain insight into virulence mechanism. As the infections are increasing rapidly, more divergence is expected followed possibly by viral adaptation. We could identify mutational hotspots which appear to be major drivers of diversity among strains, with RBD of spike protein emerging as the key region involved in interaction with ACE2 and consequently a major determinant of infection outcome. We believe that such molecular analyses correlated with clinical characteristics and host predisposition need to be evaluated at the earliest to understand viral adaptability, disease prognosis, and transmission dynamics.

positive: Virtual screening and repurposing of FDA approved drugs against COVID-19 main protease Abstract Aims In December 2019, the Coronavirus disease-2019 (COVID-19) virus has emerged in Wuhan, China. In this research, the first resolved COVID-19 crystal structure (main protease) was targeted in a virtual screening study by of FDA approved drugs dataset. In addition, a knowledge gap in relations of COVID-19 with the previously known fatal Coronaviruses (CoVs) epidemics, SARS and MERS CoVs, was covered by investigation of sequence statistics and phylogenetics. Materials and methods Molecular modeling, virtual screening, docking, sequence comparison statistics and phylogenetics of the COVID-19 main protease were investigated. Key findings COVID-19 Mpro formed a phylogenetic group with SARS CoV that was distant from MERS CoV. The identity% was 96.061 and 51.61 for COVID-19/SARS and COVID-19/MERS CoV sequence comparisons, respectively. The top 20 drugs in the virtual screening studies comprised a broad-spectrum antiviral (ribavirin), anti-hepatitis B virus (telbivudine), two vitamins (vitamin B12 and nicotinamide) and other miscellaneous systemically acting drugs. Of special interest, ribavirin had been used in treating cases of SARS CoV. Significance The present study provided a comprehensive targeting of the first resolved COVID+19 structure of Mpro and found a suitable save drugs for repurposing against the viral Mpro. Ribavirin, telbivudine, vitamin B12 and nicotinamide can be combined and used for COVID treatment. This initiative relocates already marketed and approved safe drugs for potential use in COVID-treatment.

positive: SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February-March 2020. We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.

positive: Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach The newly identified SARS-CoV-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. Spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment. In this study, 483 unique variations have been identified among the genomes of SARS-CoV-2 including 25 nonsynonymous mutations and one deletion in the spike (S) protein. Among the 26 variations detected in S, 12 variations were located at the N-terminal domain (NTD) and 6 variations at the receptor-binding domain (RBD) which might alter the interaction of S protein with the host receptor angiotensin-converting enzyme 2 (ACE2). Besides, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on results reported herein, potential inhibitors against S protein can be designed by considering these variations and their impact on protein structure.

positive: RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 ‘SARS-related-conserved’ regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 ‘SARS-CoV-2-conserved-structured’ regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5’ UTR, frame-shifting element, and 3’ UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 ‘SARS-CoV-2-conserved-unstructured’ genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

positive: A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, resulting in >300,000 reported cases worldwide as of March 21st, 2020. Here we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 in Northern California using samples from returning travelers, cruise ship passengers, and cases of community transmission with unclear infection sources. Virus genomes were sampled from 29 patients diagnosed with COVID-19 infection from Feb 3rd through Mar 15th. Phylogenetic analyses revealed at least 8 different SARS-CoV-2 lineages, suggesting multiple independent introductions of the virus into the state. Virus genomes from passengers on two consecutive excursions of the Grand Princess cruise ship clustered with those from an established epidemic in Washington State, including the WA1 genome representing the first reported case in the United States on January 19th. We also detected evidence for presumptive transmission of SARS-CoV-2 lineages from one community to another. These findings suggest that cryptic transmission of SARS-CoV-2 in Northern California to date is characterized by multiple transmission chains that originate via distinct introductions from international and interstate travel, rather than widespread community transmission of a single predominant lineage. Rapid testing and contact tracing, social distancing, and travel restrictions are measures that will help to slow SARS-CoV-2 spread in California and other regions of the USA.

positive: Multiple SARS-CoV-2 introductions shaped the early outbreak in Central Eastern Europe: comparing Hungarian data to a worldwide sequence data-matrix Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019 the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of the 21th of April, 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.

positive: Early Phylogenetic Estimate Of The Effective Reproduction Number Of 2019-nCoV To reconstruct the evolutionary dynamics of the 2019 novel coronavirus, 52 2019−nCOV genomes available on 04 February 2020 at GISAID were analysed. The two models used to estimate the reproduction number (coalescent−based exponential growth and a birth−death skyline method) indicated an estimated mean evolutionary rate of 7.8 x 10−4 subs/site/year (range 1.1x10−4−15x10−4). The estimated R value was 2.6 (range 2.1−5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.

positive: Comparative Analysis of SARS-CoV2 with Special Emphasis on Genome Sequences Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus - 2 (SARS-CoV-2), was declared a global pandemic on 11th March, 2020 by World Health Organization As of now,27th May,2020, there are about 54,88,825 infected cases and 3,49,095 deaths globally Coronavirus samples collected from all the countries have been sequenced for advanced studies in a bid to understand the structure and functioning of the virus In our study we have tried working on every available sequence to setup both comparisons and co-relations There is no such available study as of now for reference and hence it can become a pioneer stone in this direction The mortality rate calculated turns out to be 9 19%,34 37% and 6 29% for SARS-2003, MERS-2012 and COVID-19 respectively The basic reproduction rate R0 was 2-5 for SARS-2003, 0 3-0 8 for MERS-2012 and 1 4-5 7 for COVID-19 We found out the relation between number of mutations and mortality as well as phylogenetic relations High number of mutations corresponded to higher mortality rate as in countries like Italy and Spain Alpha and Beta-coronaviruses show a common ancestor from which they descended Brazil and Iran have shown similar phylogenetic descent explaining their mortality rate India however showed a distant relation from the common ancestor of other genome sequences This study highlights the mutations of the SARS-CoV2 virus as well as sets up a comparison with the previous outbreaks Similar type of studies should be conducted when more genome samples are present These results can also contribute towards making an effective anti-viral therapy and vaccines

positive: CoV3D: A database of high resolution coronavirus protein structures The SARS-CoV-2 virus is the cause of the current COVID-19 pandemic, and exemplifies the general threat to global health posed by coronaviruses. The urgent need for effective vaccines and therapies is leading to a rapid rise in the number of high resolution structures of SARS-CoV-2 proteins that collectively reveal a map of virus vulnerabilities. To assist structure-based design of vaccines and therapeutics against SARS-CoV-2 and other coronaviruses, we developed CoV3D, a database and molecular viewer of SARS-CoV-2 and other coronavirus protein structures updated weekly. CoV3D annotates structures of coronavirus proteins and their complexes with antibodies, receptors, and small molecules. Additionally, CoV3D provides information on spike glycoprotein sequence variability and polymorphisms, and maps these features onto the spike structure in the integrated molecular viewer. In order to further aid structure-based design and analysis, CoV3D includes viewable and downloadable spike glycoprotein structures with modeled glycosylation from Rosetta. CoV3D is available at: https://cov3d.ibbr.umd.edu.

positive: Variant analysis of SARS-CoV-2 strains in Middle Eastern countries Background SARS-CoV-2 is diverging from the initial Wuhan serotype, and different variants of the virus are reported. Mapping the variant strains and studying their pattern of evolution will provide better insights into the pandemic spread Methods Data on different SARS-CoV2 for WHO EMRO countries were obtained from the Chinese National Genomics Data Center (NGDC), Genbank and the Global Initiative on Sharing All Influenza Data (GISAID). Multiple sequence alignments (MSA) was performed to study the evolutionary relationship between the genomes. Variant calling, genome and variant alignment were performed to track the strains in each country. Evolutionary and phylogenetic analysis is used to explore the evolutionary hypothesis. Findings Of the total 50 samples, 4 samples did not contain any variants. Variant calling identified 379 variants. Earliest strains are found in Iranian samples. Variant alignment indicates Iran samples have a low variant frequency. Saudi Arabia has formed an outgroup. Saudi Arabia, Qatar and Kuwait were the most evolved genomes and are the countries with the highest number of cases per million. Interpretation Iran was exposed to the virus earlier than other countries in the Eastern Mediterranean Region. Funding None

positive: Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CL(pro) targeting repurposed drug candidates BACKGROUND: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. METHODS: For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CL(pro)) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. RESULTS: Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CL(pro) enzyme, which regulates viral replication machinery. CONCLUSIONS: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

positive: Binding Ability Prediction between Spike Protein and Human ACE2 Reveals the Adaptive Strategy of SARS-CoV-2 in Humans SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing an outbreak of COVID-19 globally in the past six months. A relatively higher divergence on the spike protein of SASR-CoV-2 enables it to transmit across species efficiently. We particularly believe that the adaptive mutations of the receptor-binding domain (RBD) of spike protein in SARS-CoV-2 might be essential to its high transmissibility among humans. Thus here we collected 2,142 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny, and also analyzed the interaction between the polymorphisms of spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 and coronavirus in other hosts show SARS-CoV-2 is highly possible originated from Bat-CoV (RaTG13) found in horseshoe bat and a recombination event may occur on the spike protein of Pangolin-CoV to imbue it the ability to infect humans. Moreover, compared to the S gene of SARS-CoV-2, it is more conserved in the direct-binding sites of RBD and we noticed that spike protein of SARS-CoV-2 may under a consensus evolution to adapt to human hosts better. 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 were simulated and their stability and affinity binding to hACE2 (S19-D615) were calculated. Our analysis indicates SARS-CoV-2 could infect humans from different populations with no preference, and a higher divergence in the spike protein of SARS-CoV-2 at the early stage of this pandemic may be a good indicator that could show the pathway of SARS-CoV-2 transmitting from the natural reservoir to human beings.

positive: SARS-CoV-2: Structural diversity, phylogeny, and potential animal host identification of spike glycoprotein To investigate the evolutionary history of the current pandemic outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a total of 137 genomes of coronavirus strains with release dates between January 2019 and 25 March 2020, were analyzed. To investigate the potential intermediate host of the SARS-CoV-2, we analyzed spike glycoprotein sequences from different animals, with particular emphasis on bats. We performed phylogenetic analysis and structural reconstruction of the spike glycoproteins with subsequent alignment and comparison. Our phylogenetic results revealed that SARS-CoV-2 was more similar to the bats' betacoronavirus isolates: HKU5-related from Pipistrellus abramus and HKU4-related from Tylonycteris pachypus. We also identified a yak betacoronavirus strain, YAK/HY24/CH/2017, as the closest match in the comparison of the structural models of spike glycoproteins. Interestingly, a set of unique features has been described for this particular strain of the yak betacoronavirus. Therefore, our results suggest that the human SARS-CoV-2, responsible for the current outbreak of COVID-19, could also come from yak as an intermediate host.

positive: Bioinformatic analysis indicates that SARS-CoV-2 is unrelated to known artificial coronaviruses. OBJECTIVE SARS-CoV-2 is responsible for the present coronavirus pandemic and some suggestions were made about its possible artificial origin. We, therefore, compared SARS-CoV-2 with such known viruses that were prepared in the laboratory and other relevant natural strains to estimate their genetic relatedness. MATERIALS AND METHODS BLAST and clustalW were used to identify and align viral sequences of SARS-CoV-2 to other animal coronaviruses (human, bat, mouse, pangolin) and related artificial constructs. Phylogenetics trees were then prepared using iTOL. RESULTS Our study supports the notion that known artificial coronaviruses, including the chimeric SL-SHC014-MA15 synthesized in 2015, differ too much from SARS-CoV-2 to hypothesize an artificial origin of the latter. On the contrary, our data support the natural origin of the COVID-19 virus, likely derived from bats, possibly transferred to pangolins, before spreading to man. CONCLUSIONS Speculations about the artificial origin of SARS-CoV-2 are most likely unfounded. On the contrary, when carefully handled, engineered organisms provide a unique opportunity to study biological systems in a controlled fashion. Biotechnology is a powerful tool to advance medical research and should not be abandoned because of irrational fears.

positive: High‐Coverage SARS‐CoV‐2 Genome Sequences Acquired by Target Capture Sequencing In this study, we designed a set of SARS‐CoV‐2 enrichment probes to increase the capacity for sequence‐based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS‐CoV‐2 enrichment probe set contains 502 120nt ssDNA biotin‐labeled probes designed based on all available SARS‐CoV‐2 viral sequences and it can be used to enrich for SARS‐CoV‐2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from a cell culture, three nasopharyngeal swab samples (cycle threshold [Ct] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID‐19 (positive control) and four throat swab samples from patients without COVID‐19 (negative controls), respectively. Moreover, based on these high‐quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication, and its phylogenetic relationship with other selected high‐quality samples from The Genome Variation Map (GVM). Therefore, this universal SARS‐CoV‐2 enrichment probe system can capture and enrich SARS‐CoV‐2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles. This article is protected by copyright. All rights reserved.

positive: Novel antibody epitopes dominate the antigenicity of spike glycoprotein in SARS-CoV-2 compared to SARS-CoV

positive: Tracking the Genomic Footprints of SARS-CoV-2 Transmission There is considerable public and scientific interest in the origin, spread, and evolution of SARS-CoV-2. Lu et al. recently conducted genomic sequencing and analysis of SARS-CoV-2 in Guangdong, revealing its early transmission out of Hubei and shedding light on the effectiveness of controlling local transmission chains.

positive: The genomic variation landscape of globally-circulating clades of SARS-CoV-2 defines a genetic barcoding scheme We describe fifteen major mutation events from 2,058 high-quality SARS-CoV-2 genomes deposited up to March 31st, 2020. These events define five major clades (G, I, S, D and V) of globally-circulating viral populations, representing 85.7% of all sequenced cases, which we can identify using a 10 nucleotide genetic classifier or barcode. We applied this barcode to 4,000 additional genomes deposited between March 31st and April 15th and classified successfully 95.6% of the clades demonstrating the utility of this approach. An analysis of amino acid variation in SARS-CoV-2 ORFs provided evidence of substitution events in the viral proteins involved in both host-entry and genome replication. The systematic monitoring of dynamic changes in the SARS-CoV-2 genomes of circulating virus populations over time can guide therapeutic and prophylactic strategies to manage and contain the virus and, also, with available efficacious antivirals and vaccines, aid in the monitoring of circulating genetic diversity as we proceed towards elimination of the agent. The barcode will add the necessary genetic resolution to facilitate tracking and monitoring of infection clusters to distinguish imported and indigenous cases and thereby aid public health measures seeking to interrupt transmission chains without the requirement for real-time complete genomes sequencing.

positive: COVID-19: the First Documented Coronavirus Pandemic in History Abstract The novel human coronavirus disease COVID-19 has become the fifth documented pandemic since the 1918 flu pandemic. COVID-19 was first reported in Wuhan, China, and subsequently spread worldwide. The coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses based on phylogenetic analysis. SARS-CoV-2 is believed to be a spillover of an animal coronavirus and later adapted the ability of human-to-human transmission. Because the virus is highly contagious, it rapidly spreads and continuously evolves in the human population. In this review article, we discuss the basic properties, potential origin, and evolution of the novel human coronavirus. These factors may be critical for studies of pathogenicity, antiviral designs, and vaccine development against the virus.

positive: Diagnostics and spread of SARS-CoV-2 in Western Africa: An observational laboratory-based study from Benin Information on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread in Africa is limited by fragile 2 surveillance systems and insufficient diagnostic capacity. 3 We assessed the coronavirus disease-19 (COVID-19)-related diagnostic workload in Benin, Western Africa, 4 characterized SARS-CoV-2 genomes from 12 acute cases of COVID-19, used those together with public data to 5 estimate SARS-CoV-2 transmission dynamics in a Bayesian framework, validated a widely used diagnostic dual target 6 RT-PCR kit donated to African countries, and conducted serological analyses in 68 sera from confirmed COVID-19 7 cases and from febrile patients sampled before the predicted SARS-CoV-2 introduction. 8 We found a 15-fold increase in the monthly laboratory workload due to COVID-19. Genomic surveillance showed 9 introductions of three distinct SARS-CoV-2 lineages. SARS-CoV-2 genome-based analyses yielded an R0 estimate of 10 4.4 (95% confidence interval: 2.0-7.7), suggesting intense spread of SARS-CoV-2 in Africa. RT-PCR-based tests 11 were highly sensitive but showed variation of internal controls and between diagnostic targets. Commercially available 12 SARS-CoV-2 ELISAs showed up to 25% false-positive results depending on antigen and antibody types, likely due 13 to unspecific antibody responses elicited by acute malaria according to lack of SARS-CoV-2-specific neutralizing 14 antibody responses and relatively higher parasitemia in those sera. 15 We confirm an overload of the diagnostic capacity in Benin and provide baseline information on the usability of 16 genome-based surveillance in resource-limited settings. Sero-epidemiological studies needed to assess SARS-CoV-2 17 spread may be put at stake by low specificity of tests in tropical settings globally. The increasing diagnostic challenges 18 demand continuous support of national and supranational African stakeholders.

positive: Full genome viral sequences inform patterns of SARS-CoV-2 spread into and within Israel Full genome sequences are increasingly used to track the geographic spread and transmission dynamics of viral pathogens. Here, with a focus on Israel, we sequenced 212 SARS-CoV-2 sequences and use them to perform a comprehensive analysis to trace the origins and spread of the virus. A phylogenetic analysis including thousands of globally sampled sequences allowed us to infer multiple independent introductions into Israel, followed by local transmission. Returning travelers from the U.S. contributed dramatically more to viral spread relative to their proportion in incoming infected travelers. Using phylodynamic analysis, we estimated that the basic reproduction number of the virus was initially around ~2.0-2.6, dropping by two-thirds following the implementation of social distancing measures. A comparison between reported and model-estimated case numbers indicated high levels of transmission heterogeneity in SARS-CoV-2 spread, with between 1-10% of infected individuals resulting in 80% of secondary infections. Overall, our findings underscore the ability of this virus to efficiently transmit between and within countries, as well as demonstrate the effectiveness of social distancing measures for reducing its spread.

positive: Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 10,000,000 cases and 500,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (37S and 267A) were found to exist only in its N protein, along with an extended β-hairpin in the N-Terminal Domain. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, this data will help enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.

positive: The first two cases of 2019‐nCoV in Italy: Where they come from? A novel Coronavirus, 2019‐nCoV, has been identified as the causal pathogen of an ongoing epidemic, with the first cases reported in Wuhan, China, last December 2019, and has since spread to other countries worldwide, included Europe and very recently Italy. In this short report, phylogenetic reconstruction was used to better understand the transmission dynamics of the virus from its first introduction in China focusing on the more recent evidence of infection in a couple of Chinese tourists arrived in Italy on 23rd January 2020 and labeled as Coronavirus Italian cases. A maximum clade credibility tree has been built using a dataset of 54 genome sequences of 2019‐nCoV plus two closely related bat strains (SARS‐like CoV) available in GenBank. Bayesian time‐scaled phylogenetic analysis was implemented in BEAST 1.10.4. The Bayesian phylogenetic reconstruction showed that 2019‐2020 nCoV firstly introduced in Wuhan on 25 November 2019, started epidemic transmission reaching many countries worldwide, including Europe and Italy where the two strains isolated dated back 19 January 2020, the same that the Chinese tourists arrived in Italy. Strains isolated outside China were intermixed with strains isolated in China as evidence of likely imported cases in Rome, Italy, and Europe, as well. In conclusion, this report suggests that further spread of 2019‐nCoV epidemic was supported by human mobility and that quarantine of suspected or diagnosed cases is useful to prevent further transmission. Viral genome phylogenetic analysis represents a useful tool for the evaluation of transmission dynamics and preventive action.

positive: Quantification of Intra-Host Genomic Diversity of SARS-CoV-2 Allows a High-Resolution Characterization of Viral Evolution and Reveals Functionally Convergent Variants A global cross-discipline effort is ongoing to characterize the evolution of SARS-CoV-2 virus and generate reliable epidemiological models of its diffusion. To this end, phylogenomic approaches leverage accumulating genomic mutations as barcodes to track the evolutionary history of the virus and can benefit from the surge of sequences deposited in public databases. Yet, such methods typically rely on consensus sequences representing the dominant virus lineage, whereas a complex sublineage architecture is often observed within single hosts. Furthermore, most approaches do not account for variants accumulation processes and might produce inaccurate results in condition of limited sampling, as witnessed in most countries affected by the epidemics. We introduce VERSO (Viral Evolution ReconStructiOn), a new comprehensive framework for the characterization of viral evolution and transmission from sequencing data of viral genomes. our approach accounts for accumulation of clonal mutations and uncertainty in the data, by taking advantage of the achievements of research in cancer evolution, to deliver robust phylogenomic lineage models, and exploits intra-host variant frequency profiles to characterize the sublineage similarity among samples, which may derive from uncovered infection events. The application of our approach to RNA-sequencing data of 162 SARS-CoV-2 samples generates a high-resolution model of evolution and spread, which improves recent findings on viral types and highlights the existence of patterns of co-occurrence of minor variants, revealing likely infection paths among hosts harboring the same viral lineage. The in-depth analysis of the mutational landscape of SARS-CoV-2 confirms a statistically significant increase of genomic diversity in time and identifies a number of variants that are transiting from minor to clonal state in the population. We also show that standard phylogenetic methods can produce unreliable results when handling datasets with noise and sampling limitations, as proven by the further application of VERSO to 12419 consensus sequences included in GISAID database. Notably, VERSO allows to pinpoint minor variants that might be positively or negatively selected across distinct lineages, thus driving the design of treatments and vaccines. In particular, minor variant g.29039A>U, detected in multiple viral lineages and validated on independent samples, shows that SARS-CoV-2 can lose its main Nucleocapsid immunogenic epitopes, raising concerns about the effectiveness of vaccines targeting the C-terminus of this protein. Finally, we here release the likely SARS-CoV-2 ancestral genome, obtained by resolving ambiguous SNPs that distinguish two widely-used reference genomes from human samples, by employing the Pangolin-CoV and the Bat-CoV-RaTG13 genomes. Our results show that the joint application of our framework and data-driven epidemiological models might allow to deliver a high-resolution platform for pathogen surveillance and analysis.

positive: RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881 nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into β-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370 nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.

positive: A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein SARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease—COVID-19— within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronavirus’s surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 Å. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the “closed” prefusion S structure, but is accessible in “open” conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses. HIGHLIGHTS High resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD). Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies. Structure of the SARS-COV-2 RBD in complex with antibody CR3022. Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.

positive: COVID-19 Outbreak: An Overview BACKGROUND: In late December 2019, Chinese health authorities reported an outbreak of pneumonia of unknown origin in Wuhan, Hubei Province. SUMMARY: A few days later, the genome of a novel coronavirus was released (http://viro-logical.org/t/novel-2019-coronavirus-genome/319; Wuhan-Hu-1, GenBank accession No. MN908947) and made publicly available to the scientific community. This novel coronavirus was provisionally named 2019-nCoV, now SARS-CoV-2 according to the Coronavirus Study Group of the International Committee on Taxonomy of Viruses. SARS-CoV-2 belongs to the Coronaviridae family, Betacoronavirus genus, subgenus Sarbecovirus. Since its discovery, the virus has spread globally, causing thousands of deaths and having an enormous impact on our health systems and economies. In this review, we summarize the current knowledge about the epidemiology, phylogenesis, homology modeling, and molecular diagnostics of SARS-CoV-2. Key Messages: Phylogenetic analysis is essential to understand viral evolution, whereas homology modeling is important for vaccine strategies and therapies. Highly sensitive and specific diagnostic assays are key to case identification, contact tracing, identification of the animal source, and implementation of control measures.

positive: Genome Sequence of SARS-CoV-2 Isolate Cali-01, from Colombia, Obtained Using Oxford Nanopore MinION Sequencing We report the genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a patient with symptoms of coronavirus disease 2019 (COVID-19) who was infected in Cali, Colombia. The patient had no recent travel record and did not require hospitalization. The virus genome was obtained using Oxford Nanopore MinION sequencing.

positive: Rapid whole genome sequence typing reveals multiple waves of SARS-CoV-2 spread As the pandemic SARS-CoV-2 virus has spread globally its genome has diversified to an extent that distinct clones can now be recognized, tracked, and traced. Identifying clonal groups allows for assessment of geographic spread, transmission events, and identification of new or emerging strains that may be more virulent or more transmissible. Here we present a rapid, whole genome, allele-based method (GNUVID) for assigning sequence types to sequenced isolates of SARS-CoV-2 sequences. This sequence typing scheme can be updated with new genomic information extremely rapidly, making our technique continually adaptable as databases grow. We show that our method is consistent with phylogeny and recovers waves of expansion and replacement of sequence types/clonal complexes in different geographical locations. GNUVID is available as a command line application (https://github.com/ahmedmagds/GNUVID).

positive: Distribution of COVID-19 and phylogenetic tree construction of sars-CoV-2 in Indonesia Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has spread quickly across the world and has been declared a pandemic. Indonesia has many COVID-19 cases, with a high mortality rate. This study aimed to describe the distribution of COVID-19 in Indonesia and constructed the SARS-CoV-2 phylogenetic tree from Indonesian isolates and those from other countries, including other CoVs to determine their relationship. The distribution data of COVID-19 in Indonesia were obtained from the COVID-19 Management Handling Unit and descriptively analyzed. SARS-CoV-2 isolates were retrieved from the GenBank® (National Center of Biotechnology Information, USA) and GISAID EpiCoV™ databases and were used to construct phylogenetic trees using MEGA X software. Of the 37 provinces in Indonesia, five provinces with the highest case fatality rates were DKI Jakarta, Jawa Barat, Jawa Timur, and Banten, and the five provinces with the highest cure rate were Kepulauan Riau, Bali, Aceh, Gorontalo, and DI Yogyakarta. SARS-CoV-2 Indonesian isolates were closely related to SARS-CoV-2 isolates from other countries. The rapid and widespread distribution of SARS-CoV-2 in Indonesia was caused by the lack of compliance with territorial restrictions and dishonesty with medical personnel. These data revealed that mutations can occur during the transmission process, which can be caused by a history of travel and increased patient immunity.

positive: Novel coronavirus takes flight from bats? Two recent studies provide initial insights into a novel coronavirus that is associated with an outbreak of human respiratory disease.

positive: Introductions and early spread of SARS-CoV-2 in France, 24 January to 23 March 2020 Following SARS-CoV-2 emergence in China, a specific surveillance was implemented in France. Phylogenetic analysis of sequences retrieved through this surveillance suggests that detected initial introductions, involving non-clade G viruses, did not seed local transmission. Nevertheless, identification of clade G variants subsequently circulating in the country, with the earliest from a patient who neither travelled to risk areas nor had contact with travellers, suggests that SARS-CoV-2 might have been present before the first recorded local cases.

positive: Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Viruses Reveal Mosaic Pattern of Phylogeographical Distribution The Coronavirus Disease-2019 (COVID-19) that started in Wuhan, China in December 2019 has spread worldwide emerging as a global pandemic. The severe respiratory pneumonia caused by the novel SARS-CoV-2 has so far claimed more than 60,000 lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 displays high transmission rates, their underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nsp13-16 (ORF1b polyprotein) and S-Protein within 10 viral isolates from the USA. These viral proteins are involved in RNA replication, indicating highly evolved viral strains circulating in the population of USA than other countries. Furthermore, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of the USA isolate (MT188341) leading to shift in amino acid frame from position 2540 onwards. Through the construction of SARS-CoV-2-human interactome, we further revealed that multiple host proteins (PHB, PPP1CA, TGF-β, SOCS3, STAT3, JAK1/2, SMAD3, BCL2, CAV1 & SPECC1) are manipulated by the viral proteins (nsp2, PL-PRO, N-protein, ORF7a, M-S-ORF3a complex, nsp7-nsp8-nsp9-RdRp complex) for mediating host immune evasion. Thus, the replicative machinery of SARS-CoV-2 is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies.

positive: Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus Recently, a novel coronavirus (2019-nCoV) has emerged from Wuhan, China, causing symptoms in humans similar to those caused by severe acute respiratory syndrome coronavirus (SARS-CoV). Since the SARS-CoV outbreak in 2002, extensive structural analyses have revealed key atomic-level interactions between the SARS-CoV spike protein receptor-binding domain (RBD) and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. Here, we analyzed the potential receptor usage by 2019-nCoV, based on the rich knowledge about SARS-CoV and the newly released sequence of 2019-nCoV. First, the sequence of 2019-nCoV RBD, including its receptor-binding motif (RBM) that directly contacts ACE2, is similar to that of SARS-CoV, strongly suggesting that 2019-nCoV uses ACE2 as its receptor. Second, several critical residues in 2019-nCoV RBM (particularly Gln493) provide favorable interactions with human ACE2, consistent with 2019-nCoV's capacity for human cell infection. Third, several other critical residues in 2019-nCoV RBM (particularly Asn501) are compatible with, but not ideal for, binding human ACE2, suggesting that 2019-nCoV has acquired some capacity for human-to-human transmission. Last, while phylogenetic analysis indicates a bat origin of 2019-nCoV, 2019-nCoV also potentially recognizes ACE2 from a diversity of animal species (except mice and rats), implicating these animal species as possible intermediate hosts or animal models for 2019-nCoV infections. These analyses provide insights into the receptor usage, cell entry, host cell infectivity and animal origin of 2019-nCoV and may help epidemic surveillance and preventive measures against 2019-nCoV.IMPORTANCE The recent emergence of Wuhan coronavirus (2019-nCoV) puts the world on alert. 2019-nCoV is reminiscent of the SARS-CoV outbreak in 2002 to 2003. Our decade-long structural studies on the receptor recognition by SARS-CoV have identified key interactions between SARS-CoV spike protein and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. One of the goals of SARS-CoV research was to build an atomic-level iterative framework of virus-receptor interactions to facilitate epidemic surveillance, predict species-specific receptor usage, and identify potential animal hosts and animal models of viruses. Based on the sequence of 2019-nCoV spike protein, we apply this predictive framework to provide novel insights into the receptor usage and likely host range of 2019-nCoV. This study provides a robust test of this reiterative framework, providing the basic, translational, and public health research communities with predictive insights that may help study and battle this novel 2019-nCoV.

positive: Phylogenetic Analysis of the Novel Coronavirus Reveals Important Variants in Indian Strains Recently classified as a pandemic by WHO, novel Corononavirus 2019 has affected almost every corner of the globe causing human deaths in a range of hundred thousands. The virus having its roots in Wuhan (China) has been spread over the world by its own property to change itself accordingly. These changes correspond to its transmission and pathogenicity due to which the concept of social distancing appeared into the picture. In this paper, a few findings from the whole genome sequence analysis of viral genome sequences submitted from India are presented. The data used for analysis comprises 440 collective genome sequences of virus submitted in GenBank, GISAID, and SRA projects, from around the world as well as 28 viral sequences from India. Multiple sequence alignment of all genome sequences was performed and analysed. A novel non-synonymous mutation 4809C>T (S1515F) in NSP3 gene of SARS-CoV2 Indian strains is reported along with other frequent and important changes from around the world: 3037C>T, 14408C>T, and 23403A>G. The novel change was observed in samples collected in the month of March, whereas was found to be absent in samples collected in January with the respective persons’ travel history to China. Phylogenetic analysis clustered the sequences with this change as one separate clade. Mutation was predicted as stabilising change by insilco tool DynaMut. A second patient in the world to our knowledge with multiple (Wuhan and USA) strain contraction was observed in this study. The infected person is among the two early infected patients with travel history to China. Strains sequenced in Iran stood out to have different variants, as most of the reported frequent variants were not observed. The objective of this paper is to highlight the similarities and changes observed in the submitted Indian viral strains. This helps to keep track on the activity, that how virus is changing into a new subtype. Major strains observed were European with the novel change in India and other being emergent clade of Iran. Its important to observe the changes in NSP3 gene, as this gene has been reported with extensive positive selection as well as potential drug target. Extensive Positive Selection Drives the Evolution of Nonstructural Proteins. With the limited number of sequences this was the only frequent novel non-synonymous change observed from Indian strains, thereby making this change vulnerable for investigation in future. This paper has a special focus on tracking of Indian viral sequences submitted in public domain.

positive: Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design Novel coronavirus has wrecked medical and health care facilities claiming ~5% death tolls globally. All efforts to contain the pathogenesis either using inhibitory drugs or vaccines largely remained futile due to a lack of better understanding of the genomic feature of this virus. In the present study, we compared the 2019-nCoV with other coronaviruses, which indicated that bat-SARS like coronavirus could be a probable ancestor of the novel coronavirus. The protein sequence similarity of pangolin-hCoV and bat-hCoV with human coronavirus was higher as compared to their nucleotide similarity denoting the occurrence of more synonymous mutations in the genome. Phylogenetic and alignment analysis of 591 novel coronaviruses of different clades from Group I to Group V revealed several mutations and concomitant amino acid changes. Detailed investigation on nucleotide substitution unfolded 100 substitutions in the coding region of which 43 were synonymous and 57 were of non-synonymous type. The non-synonymous substitutions resulting into 57 amino acid changes were found to be distributed over different hCoV proteins with maximum on spike protein. An important diamino acid change RG to KR was observed in ORF9 protein. Additionally, several interesting features of the novel coronavirus genome have been highlighted in respect to various other human infecting viruses which may explain extreme pathogenicity, infectivity and simultaneously the reason behind failure of the antiviral therapies. Summary This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. This analysis will provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.

positive: Unraveling virus relationships by structure-based phylogenetic classification Delineation of the intricacies of protein function from macromolecular structure constitutes a continual obstacle in the study of cell and pathogen biology. Structure-based phylogenetic analysis has emerged as a powerful tool for addressing this challenge, allowing the detection and quantification of conserved architectural properties between proteins, including those with low or no detectable sequence homology. With a focus on viral protein structure, we highlight how a number of investigations have utilized this powerful method to infer common functionality and ancestry.

positive: An exploration of the SARS-CoV-2 spike receptor binding domain (RBD) – a complex palette of evolutionary and structural features SARS-CoV-2 spike protein (S) is associated with the entry of virus inside the host cell by recruiting its loop dominant receptor binding domain (RBD) and interacting with the host ACE2 receptor. Our study deploying a two-tier approach encompassing evolutionary and structural analysis provides a comprehensive picture of the RBD, which could be of potential use for better understanding the RBD and address its druggability issues. Resorting to an ensemble of sequence space exploratory tools including co-evolutionary analysis and deep mutational scans we provide a quantitative insight into the evolutionarily constrained subspace of the RBD sequence space. Guided by structure network analysis and Monte Carlo simulation we highlight regions inside the RBD, which are critical for providing structural integrity and conformational flexibility of the binding cleft. We further deployed fuzzy C-means clustering by plugging the evolutionary and structural features of discrete structure blocks of RBD to understand which structure blocks share maximum overlap based on their evolutionary and structural features. Deploying this multi-tier interlinked approach, which essentially distilled the evolutionary and structural features of RBD, we highlight discrete region, which could be a potential druggable pocket thereby destabilizing the structure and addressing evolutionary routes.

positive: Genome-wide data inferring the evolution and population demography of the novel pneumonia coronavirus (SARS-CoV-2) Since December 2019, coronavirus disease 2019 (COVID-19) emerged in Wuhan, Central China and rapidly spread throughout China. Up to March 3, 2020, SARS-CoV-2 has infected more than 89,000 people in China and other 66 countries across six continents. In this study, we used 10 new sequenced genomes of SARS-CoV-2 and combined 136 genomes from GISAID database to investigate the genetic variation and population demography through different analysis approaches (e.g. Network, EBSP, Mismatch, and neutrality tests). The results showed that 80 haplotypes had 183 substitution sites, including 27 parsimony-informative and 156 singletons. Sliding window analyses of genetic diversity suggested a certain mutations abundance in the genomes of SARS-CoV-2, which may be explaining the existing widespread and high adaptation of the deadly virus. Phylogenetic analysis showed that the view, pangolin acted as an intermediate host, may be controversial. The network indicated that, in the original haplotype (H14), one patient sample lived near the Huanan seafood market (approximate 2 km), indicating high possibility of the patient having a history of unconscious contact with this market. However, based on this clue, we cannot accurately concluded that whether this market was the origin center of SARS-CoV-2. Additionally, 16 genomes, collected from this market, assigned to 10 haplotypes, indicated a circulating infection within the market in a short term and then leading to the outbreak of SARS-CoV-2 in Wuhan and other areas. The EBSP results showed that the first estimated expansion date of SARS-CoV-2 began from 7 December 2019, which may indicated that the transmission could have begun from person to person in mid to late November.

positive: The global spread of 2019-nCoV: a molecular evolutionary analysis The global spread of the 2019-nCoV is continuing and is fast moving, as indicated by the WHO raising the risk assessment to high. In this article, we provide a preliminary phylodynamic and phylogeographic analysis of this new virus. A Maximum Clade Credibility tree has been built using the 29 available whole genome sequences of 2019-nCoV and two whole genome sequences that are highly similar sequences from Bat SARS-like Coronavirus available in GeneBank. We are able to clarify the mechanism of transmission among the countries which have provided the 2019-nCoV sequence isolates from their patients. The Bayesian phylogeographic reconstruction shows that the 2019-2020 nCoV most probably originated from the Bat SARS-like Coronavirus circulating in the Rhinolophus bat family. In agreement with epidemiological observations, the most likely geographic origin of the new outbreak was the city of Wuhan, China, where 2019-nCoV time of the most recent common ancestor emerged, according to molecular clock analysis, around November 25th, 2019. These results, together with previously recorded epidemics, suggest a recurring pattern of periodical epizootic outbreaks due to Betacoronavirus. Moreover, our study describes the same population genetic dynamic underlying the SARS 2003 epidemic, and suggests the urgent need for the development of effective molecular surveillance strategies of Betacoronavirus among animals and Rhinolophus of the bat family.

positive: [Analysis of variation and evolution of SARS-CoV-2 genome] OBJECTIVE: To analyze the evolution and variation of SARS-CoV-2 during the epidemic starting at the end of 2019. METHODS: We downloaded the full-length genome sequence of SARS-CoV-2 from the databases of GISAID and NCBI. Using the software for bioinformatics including MEGA-X, BEAST, and TempEst, we constructed the genomic evolution tree, inferred the time evolution signal of the virus, calculated the tMRCA time of the virus and analyzed the selection pressure of the virus during evolution. RESULTS: The phylogenetic tree showed that SARS-CoV-2 belonged to the Sarbecovirus subgenus of ß Coronavirus genus together with bat coronavirus BetaCoV/bat/Yunnan/RaTG13/2013, bat-SL-CoVZC45, bat-SL-CoVZXC21 and SARS-CoV. The genomic sequences of SARS-CoV-2 isolated from the ongoing epidemic showed a weak time evolution signal with an average tMRCA time of 73 days (95% CI: 38.9-119.3 days). No positive time evolution signal was found between SARS-CoV-2 and BetaCoV/bat/Yunnan/RaTG13/2013, but the former virus had a strong positive temporal evolution relationship with bat-SL-CoVZC45 and SARS-CoV. The major cause for mutations of SARS-CoV-2 was the pressure of purification selection during the epidemic. CONCLUSIONS: SARS-CoV-2 may have emerged as early as November, 2019, originating most likely from bat-associated coronavirus. This finding may provide evidence for tracing the sources and evolution of the virus.

positive: Deep phylogenetic analysis of Orthocoronavirinae genomes traces the origin, evolution and transmission route of 2019 novel coronavirus The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan city, China in December 2019 and thereafter its spillover across the world has created a global pandemic and public health crisis. Today, it appears as a threat to human civilization. Scientists and medical practitioners across the world are involved to trace out the origin and evolution of SARS-CoV-2 (also called 2019 novel coronavirus and referred as 2019-nCoV), its transmission route, cause of pathogenicity, and possible remedial action. In this work, we aim to find out the origin, evolutionary patternthat led to its pathogenicity and possible transmission pathway of 2019-nCoV. To achieve the aims we conducted a large-scale deep phylogenetic analysis on the 162 complete Orthocoronavirinae genomes consisting of four genera namely Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus, their gene trees analysis and subsequently genome and gene recombination analyses. Our analyses revealed that i) bat, pangolin and anteater are the natural hosts of 2019-nCoV, ii) outbreak of 2019-nCoV took place via inter-intra species transmission mode, iii) host-specific adaptive mutation made 2019-nCoV more virulent, and the presence of widespread recombination events led to the evolution of new 2019-nCoV strain and/or could be determinant of its pathogenicity. Highlights Orthocoronavirinae genome phylogeny revealed that bat, pangolin and anteater are natural reservoir hosts of novel coronavirus (2019-nCoV/SARS-CoV-2). Host-specific adaptive mutation occurred among the coronaviruses. Transmission of 2019-nCoV to human took place by inter-intra species mode of transmission. Presence of widespread recombination events led to the evolution of new 2019-nCoV strain and/or could be determinant of its pathogenicity.

positive: Evidence of the Recombinant Origin and Ongoing Mutations in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) The recent global outbreak of viral pneumonia designated as Coronavirus Disease 2019 (COVID-19) by coronavirus (SARS-CoV-2) has threatened global public health and urged to investigate its source. Whole genome analysis of SARS-CoV-2 revealed ~96% genomic similarity with bat CoV (RaTG13) and clustered together in phylogenetic tree. Furthermore, RaTGl3 also showed 97.43% spike protein similarity with SARS-CoV-2 suggesting that RaTGl3 is the closest strain. However, RBD and key amino acid residues supposed to be crucial for human-to-human and cross-species transmission are homologues between SARS-CoV-2 and pangolin CoVs. These results from our analysis suggest that SARS-CoV-2 is a recombinant virus of bat and pangolin CoVs. Moreover, this study also reports mutations in coding regions of 125 SARS-CoV-2 genomes signifying its aptitude for evolution. In short, our findings propose that homologous recombination has been occurred between bat and pangolin CoVs that triggered cross-species transmission and emergence of SARS-CoV-2, and, during the ongoing outbreak, SARS-CoV-2 is still evolving for its adaptability.

positive: Early phylogenetic estimate of the effective reproduction number of SARS‐CoV‐2 To reconstruct the evolutionary dynamics of the 2019 novel‐coronavirus recently causing an outbreak in Wuhan, China, 52 SARS‐CoV‐2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent‐based exponential growth and a birth‐death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10(−4) subs/site/year (range, 1.1 × 10(−4)‐15 × 10(−4)) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1‐5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.

positive: Introductions and early spread of SARS-CoV-2 in the New York City area New York City (NYC) has emerged as one of the epicenters of the current SARS-CoV-2 pandemic. To identify the early transmission events underlying the rapid spread of the virus in the NYC metropolitan area, we sequenced the virus causing COVID-19 in patients seeking care at the Mount Sinai Health System. Phylogenetic analysis of 84 distinct SARS-CoV2 genomes indicates multiple, independent but isolated introductions mainly from Europe and other parts of the United States. Moreover, we find evidence for community transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different neighborhoods of the city.

positive: The establishment of reference sequence for SARS-CoV-2 and variation analysis Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.

positive: Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features. Twelve complete genomes of three novel coronaviruses-bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d)-were sequenced. Comparative genome analysis showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also observed. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene has been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further molecular epidemiological studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.

positive: Importation of SARS-CoV-2 infection leads to major COVID-19 epidemic in Taiwan OBJECTIVE: COVID-19 has recently become a pandemic affecting many countries worldwide. This study aims to evaluate the current status of COVID-19 in Taiwan and analyze the source of infection. METHODS: National data regarding SARS-CoV-2 infection were obtained from Taiwan. CDC at the end of April 2020. These data were subjected to analysis of the current status and correlation between indigenous and imported COVID-19 cases. A phylogenetic tree was created to analyze the phylogeny of Taiwanese SARS-CoV-2 isolates. RESULTS: The first case of SARS-CoV-2 infection in Taiwan was detected on January 21, 2020. Epidemiological data indicate that by April 30, there were a total of 429 COVID-19 confirmed cases with the death rate of 1.3%. Most cases were identified as imported (79.9%; 343/429), with the majority originating from the United States of America (22.1%) and the United Kingdom (17.6%). Results from phylogenetic tree analyses indicate that the Taiwanese SARS-CoV-2 isolates were clustered with the SARS-CoV-2 isolates from other countries (bootstrap value 98%) and sub-clustered with bat SARS-like coronaviruses (bootstrap value 99%). CONCLUSION: This study suggests that the importation of SARS-CoV-2 infection was the primary risk-factor resulting in the COVID-19 epidemic in Taiwan.

positive: Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection Lack of effective treatment strategy and vaccine makes SARS-CoV-2 infection a big threat to mankind. Analyzing the host transcriptional changes in response to virus infection will help delineate the biological processes impacted by the virus and will potentially facilitate drug development. Using RNA seq datasets of virus infected lung cell lines A549 (infected with either SARS-CoV-2 or Influenza A virus (IAV)) and Calu3 (infected with either SARS-CoV-2 or MERS-CoV), we present a detailed analysis of genes expression changes in response to each of these viral infections. Upregulation of the antiviral interferon signaling was observed with all three viral infections. However, upregulation of the cytokine/inflammatory processes, downregulation of mitochondrial organization and respiration processes, and perturbation in the autophagic processes were specifically observed in SARS-CoV-2 infected cells, which were absent in IAV infected cells. Upregulation of the inflammatory processes was concordant with the gene expression signature of COVID-19 lungs and with inflammatory symptoms observed in severe cases of COVID-19 patients. Coexpression networks analysis also facilitated the identification of protein-protein interaction (PPI) subnetworks of genes in the inflammation and mitochondrial processes that were either coordinately up or downregulated in SARS-CoV-2 infected cells, respectively. Comparing the expression of marker genes of lung cell types from single cell RNA seq data with expression profile of A549 cells revealed that they likely represent the lung epithelial lineage cells. The cellular processes uniquely perturbed in infected cells that were identified in this analysis likely delineates lung epithelial cells response to the SARS-CoV-2 infection.

positive: First detection and genome sequencing of SARS-CoV-2 in an infected cat in France After its first description in Wuhan (China), SARS-CoV-2 the agent of coronavirus disease 2019 (COVID-19) rapidly spread worldwide. Previous studies suggested that pets could be susceptible to SARS-CoV-2. Here, we investigated the putative infection by SARS-CoV-2 in 22 cats and 11 dogs from owners previously infected or suspected of being infected by SARS-CoV-2. For each animal, rectal, nasopharyngeal swabs and serum were taken. Swabs were submitted to RT-qPCR assays targeting 2 genes of SARS-CoV-2. All dogs were tested SARS-CoV-2 negative. One cat was tested positive by RT-qPCR on rectal swab. Nasopharyngeal swabs from this animal were tested negative. This cat showed mild respiratory and digestive signs. Serological analysis confirms the presence of antibodies against the SARS-CoV-2 in both serum samples taken 10 days apart. Genome sequence analysis revealed that the cat SARS-CoV-2 belongs to the phylogenetic clade A2a like most of the French human SARS-CoV-2. This study reports for the first time the natural infection of a cat in France (near Paris) probably through their owners. There is currently no evidence that cats can spread COVID-19 and owners should not abandon their pets or compromise their welfare.

positive: The UCSC SARS-CoV-2 Genome Browser Background Researchers are generating molecular data pertaining to the SARS-CoV-2 RNA genome and its proteins at an unprecedented rate during the COVID-19 pandemic. As a result, there is a critical need for rapid and continuously updated access to the latest molecular data in a format in which all data can be quickly cross-referenced and compared. We adapted our genome browser visualization tool to the viral genome for this purpose. Molecular data, curated from published studies or from database submissions, are mapped to the viral genome and grouped together into “annotation tracks” where they can be visualized along the linear map of the viral genome sequence and programmatically downloaded in standard format for analysis. Results The UCSC Genome Browser for SARS-CoV-2 (https://genome.ucsc.edu/covid19.html) provides continuously updated access to the mutations in the many thousands of SARS-CoV-2 genomes deposited in GISAID and the international nucleotide sequencing databases, displayed alongside phylogenetic trees. These data are augmented with alignments of bat, pangolin, and other animal and human coronavirus genomes, including per-base evolutionary rate analysis. All available annotations are cross-referenced on the virus genome, including those from major databases (PDB, RFAM, IEDB, UniProt) as well as up-to-date individual results from preprints. Annotated data include predicted and validated immune epitopes, promising antibodies, RT-PCR and sequencing primers, CRISPR guides (from research, diagnostics, vaccines, and therapies), and points of interaction between human and viral genes. As a community resource, any user can add manual annotations which are quality checked and shared publicly on the browser the next day. Conclusions We invite all investigators to contribute additional data and annotations to this resource to accelerate research and development activities globally. Contact us at genome-www@soe.ucsc.edu with data suggestions or requests for support for adding data. Rapid sharing of data will accelerate SARS-CoV-2 research, especially when researchers take time to integrate their data with those from other labs on a widely-used community browser platform with standardized machine-readable data formats, such as the SARS-CoV-2 Genome Browser.

positive: Amino acid variation analysis of surface spike glycoprotein at 614 in SARS-CoV-2 strains Abstract As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to disperse globally with worrisome speed, identifying amino acid variations in the virus could help to understand the characteristics of it. Here, we studied 489 SARS-CoV-2 genomes obtained from 32 countries from the Nextstrain database and performed phylogenetic tree analysis by clade, country, and genotype of the surface spike glycoprotein (S protein) at site 614. We found that virus strains from mainland China were mostly distributed in Clade B and Clade undefined in the phylogenetic tree, with very few found in Clade A. In contrast, Clades A2 (one case) and A2a (112 cases) predominantly contained strains from European regions. Moreover, Clades A2 and A2a differed significantly from those of mainland China in age of infected population (P = 0.0071, mean age 40.24 to 46.66), although such differences did not exist between the US and mainland China. Further analysis demonstrated that the variation of the S protein at site 614 (QHD43416.1: p.614D>G) was a characteristic of stains in Clades A2 and A2a. Importantly, this variation was predicted to have neutral or benign effects on the function of the S protein. In addition, global quality estimates and 3D protein structures tended to be different between the two S proteins. In summary, we identified different genomic epidemiology among SARS-CoV-2 strains in different clades, especially in an amino acid variation of the S protein at 614, revealing potential viral genome divergence in SARS-CoV-2 strains.

positive: A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster BACKGROUND: An ongoing outbreak of pneumonia associated with a novel coronavirus was reported in Wuhan city, Hubei province, China. Affected patients were geographically linked with a local wet market as a potential source. No data on person-to-person or nosocomial transmission have been published to date. METHODS: In this study, we report the epidemiological, clinical, laboratory, radiological, and microbiological findings of five patients in a family cluster who presented with unexplained pneumonia after returning to Shenzhen, Guangdong province, China, after a visit to Wuhan, and an additional family member who did not travel to Wuhan. Phylogenetic analysis of genetic sequences from these patients were done. FINDINGS: From Jan 10, 2020, we enrolled a family of six patients who travelled to Wuhan from Shenzhen between Dec 29, 2019 and Jan 4, 2020. Of six family members who travelled to Wuhan, five were identified as infected with the novel coronavirus. Additionally, one family member, who did not travel to Wuhan, became infected with the virus after several days of contact with four of the family members. None of the family members had contacts with Wuhan markets or animals, although two had visited a Wuhan hospital. Five family members (aged 36-66 years) presented with fever, upper or lower respiratory tract symptoms, or diarrhoea, or a combination of these 3-6 days after exposure. They presented to our hospital (The University of Hong Kong-Shenzhen Hospital, Shenzhen) 6-10 days after symptom onset. They and one asymptomatic child (aged 10 years) had radiological ground-glass lung opacities. Older patients (aged >60 years) had more systemic symptoms, extensive radiological ground-glass lung changes, lymphopenia, thrombocytopenia, and increased C-reactive protein and lactate dehydrogenase levels. The nasopharyngeal or throat swabs of these six patients were negative for known respiratory microbes by point-of-care multiplex RT-PCR, but five patients (four adults and the child) were RT-PCR positive for genes encoding the internal RNA-dependent RNA polymerase and surface Spike protein of this novel coronavirus, which were confirmed by Sanger sequencing. Phylogenetic analysis of these five patients' RT-PCR amplicons and two full genomes by next-generation sequencing showed that this is a novel coronavirus, which is closest to the bat severe acute respiatory syndrome (SARS)-related coronaviruses found in Chinese horseshoe bats. INTERPRETATION: Our findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings, and the reports of infected travellers in other geographical regions. FUNDING: The Shaw Foundation Hong Kong, Michael Seak-Kan Tong, Respiratory Viral Research Foundation Limited, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, Sanming Project of Medicine (Shenzhen), and High Level-Hospital Program (Guangdong Health Commission).

positive: Polymorphism and selection pressure of SARS-CoV-2 vaccine and diagnostic antigens: implications for immune evasion and serologic diagnostic performance The ongoing SARS-CoV-2 pandemic has triggered multiple efforts for serological tests and vaccine development. Most of these tests and vaccines are based on the Spike glycoprotein (S) or the Nucleocapsid (N) viral protein. Conservation of these antigens among viral strains is critical to ensure optimum diagnostic test performance and broad protective efficacy, respectively. We assessed N and S antigen diversity from 17,853 SARS-CoV-2 genome sequences and evaluated selection pressure. Up to 6-7 incipient phylogenetic clades were identified for both antigens, confirming early variants of the S antigen and identifying new ones. Significant diversifying selection was detected at multiple sites for both antigens. Some sequence variants have already spread in multiple regions, in spite of their low frequency. In conclusion, the N and S antigens of SARS-CoV-2 are well conserved antigens, but new clades are emerging and may need to be included in future diagnostic and vaccine formulations.

positive: Betacoronavirus Genomes: How Genomic Information has been Used to Deal with Past Outbreaks and the COVID-19 Pandemic In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of coronaviruses in the aftermath of the 2002-2003 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-2020 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2. We focus on shared genomic features of the betacoronaviruses and the application of genomic information to phylogenetic analysis, molecular epidemiology and the design of diagnostic systems, potential drugs and vaccine candidates.

positive: Conserved Genomic Terminals of SARS-CoV-2 as Co-evolving Functional Elements and Potential Therapeutic Targets To identify features in the genome of the SARS-CoV-2 pathogen responsible for the COVID-19 pandemic that may contribute to its viral replication, host pathogenicity, and vulnerabilities, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from other well-characterized human and animal coronavirus genomes. Our analyses suggest the presence of unique sequence signatures in the 3’-untranslated region (UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2, SARS-CoV, as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host microRNA-mediated interaction between the 3’-UTR and human microRNA hsa-miR-1307-3p based on predicted, yet extensive, complementary base-pairings and similar interactions involving the Influenza A H1N1 virus. This interaction also suggests a possible survival mechanism, whereby a mutation in the SARS-CoV-2 3’-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection, and the exploitation of conserved features in the 3’-UTR as therapeutic targets warrant further investigation.

positive: Temporal signal and the phylodynamic threshold of SARS-CoV-2 The ongoing SARS-CoV-2 outbreak marks the first time that large amounts of genome sequence data have been generated and made publicly available in near real-time. Early analyses of these data revealed low sequence variation, a finding that is consistent with a recently emerging outbreak, but which raises the question of whether such data are sufficiently informative for phylogenetic inferences of evolutionary rates and time scales. The phylodynamic threshold is a key concept that refers to the point in time at which sufficient molecular evolutionary change has accumulated in available genome samples to obtain robust phylodynamic estimates. For example, before the phylodynamic threshold is reached, genomic variation is so low that even large amounts of genome sequences may be insufficient to estimate the virus’s evolutionary rate and the time scale of an outbreak. We collected genome sequences of SARS-CoV-2 from public databases at 8 different points in time and conducted a range of tests of temporal signal to determine if and when the phylodynamic threshold was reached, and the range of inferences that could be reliably drawn from these data. Our results indicate that by February 2nd 2020, estimates of evolutionary rates and time scales had become possible. Analyses of subsequent data sets, that included between 47 to 122 genomes, converged at an evolutionary rate of about 1.1×10−3 subs/site/year and a time of origin of around late November 2019. Our study provides guidelines to assess the phylodynamic threshold and demonstrates that establishing this threshold constitutes a fundamental step for understanding the power and limitations of early data in outbreak genome surveillance.

positive: Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes Genome Detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present The Genome Detective Coronavirus Typing Tool that can accurately identify novel coronavirus (2019-nCoV) sequences isolated in China and around the world. The tool can accept up to 2,000 sequences per submission and the analysis of a new whole genome sequence will take approximately one minute. The tool has been tested and validated with hundreds of whole genomes from ten coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for 2019-nCoV. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines.

positive: Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2 / HCoV-19) using whole genomic data The outbreak of COVID-19 started in mid-December 2019 in Wuhan, China. Up to 29 February 2020, SARS-CoV-2 (HCoV-19 / 2019-nCoV) had infected more than 85 000 people in the world. In this study, we used 93 complete genomes of SARS-CoV-2 from the GISAID EpiFlu TM database to investigate the evolution and human-to-human transmissions of SARS-CoV-2 in the first two months of the outbreak. We constructed haplotypes of the SARS-CoV-2 genomes, performed phylogenomic analyses and estimated the potential population size changes of the virus. The date of population expansion was calculated based on the expansion parameter tau ( τ) using the formula t= τ/2 u. A total of 120 substitution sites with 119 codons, including 79 non-synonymous and 40 synonymous substitutions, were found in eight coding-regions in the SARS-CoV-2 genomes. Forty non-synonymous substitutions are potentially associated with virus adaptation. No combinations were detected. The 58 haplotypes (31 found in samples from China and 31 from outside China) were identified in 93 viral genomes under study and could be classified into five groups. By applying the reported bat coronavirus genome (bat-RaTG13-CoV) as the outgroup, we found that haplotypes H13 and H38 might be considered as ancestral haplotypes, and later H1 was derived from the intermediate haplotype H3. The population size of the SARS-CoV-2 was estimated to have undergone a recent expansion on 06 January 2020, and an early expansion on 08 December 2019. Furthermore, phyloepidemiologic approaches have recovered specific directions of human-to-human transmissions and the potential sources for international infected cases.

positive: Genome Analysis of SARS-CoV-2 Isolate from Bangladesh Recently the first genome sequence for a Severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 isolate from Bangladesh became available. The sequencing was carried out by the Child Health Research Foundation and provided the first insight into the genetic details of the viral strain responsible for the SARS-CoV-2 infections in Bangladesh. Here we carried out a comparative study were we explored the phylogenetic relationship between the Bangladeshi isolate with other isolates from different parts of the world. Afterwards we identified single nucleotide variants in the Bangladeshi isolate, using the Wuhan virus reference sequence. We found a total of 9 variants in the Bangladeshi isolate using 2 separate tools. Barring 2, the rest of these variants were also observed in other isolates from different countries. Most of the variants occurred in the ORF1ab gen. Another noteworthy finding was a sequence of three consecutive variants in the N protein gene that were observed in other isolates as well. Lastly the phylogenetic analysis revealed a close relationship between the Bangladeshi isolate and those from Taiwan, Kazakhstan, Greece, California, Spain, Israel, and Sri Lanka.

positive: In silico prediction of potential inhibitors for the Main protease of SARS-CoV-2 using molecular docking and dynamics simulation based drug-repurposing BACKGROUND: The rapidly enlarging COVID-19 pandemic caused by the novel SARS-corona virus-2 is a global public health emergency of an unprecedented level. Unfortunately no treatment therapy or vaccine is yet available to counter the SARS-CoV-2 infection, which substantiates the need to expand research efforts in this direction. The indispensable function of the main protease in virus replication makes this enzyme a promising target for inhibitors screening and drug discovery to treat novel coronavirus infection. The recently concluded α-ketoamide ligand-bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al. has revealed the potential inhibitor binding mechanism and the molecular determinants responsible for substrate binding. METHODS: For the study, we have targeted the SARS-CoV-2 Mpro for the screening of FDA approved antiviral drugs and carried out molecular docking based virtual screening. Further molecular dynamic simulation studies of the top three selected drugs carried out to investigated for their binding affinity and stability in the SARS-CoV-2 Mpro active site. The phylogenetic analysis was also performed to know the relatedness between the SARS-CoV-2 genomes isolated from different countries. RESULTS: The phylogenetic analysis of the SARS-CoV-2 genome reveals that the virus is closely related to the Bat-SL-CoV and does not exhibit any divergence at the genomic level. Molecular docking studies revealed that among the 77 drugs, screened top ten drugs shows good binding affinities, whereas the top three drugs: Lopinavir-Ritonavir, Tipranavir, and Raltegravir were undergone for molecular dynamics simulation studies for their conformational stability in the active site of the SARS-CoV-2 Mpro protein. CONCLUSIONS: In the present study among the library of FDA approved antiviral drugs, the top three inhibitors Lopinavir-Ritonavir, Tipranavir, and Raltegravir show the best molecular interaction with the main protease of SARS-CoV-2. However, the in-vitro efficacy of the drug molecules screened in this study further needs to be corroborated by carrying out a biochemical and structural investigation.

positive: Introductions and early spread of SARS-CoV-2 in France Following the emergence of coronavirus disease (COVID-19) in Wuhan, China in December 2019, specific COVID-19 surveillance was launched in France on January 10, 2020. Two weeks later, the first three imported cases of COVID-19 into Europe were diagnosed in France. We sequenced 97 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from samples collected between January 24 and March 24, 2020 from infected patients in France. Phylogenetic analysis identified several early independent SARS-CoV-2 introductions without local transmission, highlighting the efficacy of the measures taken to prevent virus spread from symptomatic cases. In parallel, our genomic data reveals the later predominant circulation of a major clade in many French regions, and implies local circulation of the virus in undocumented infections prior to the wave of COVID-19 cases. This study emphasizes the importance of continuous and geographically broad genomic sequencing and calls for further efforts with inclusion of asymptomatic infections.

positive: Phylogenetic study of 2019-nCoV by using alignment-free method The origin and early spread of 2019-nCoV is studied by phylogenetic analysis using IC-PIC alignment-free method based on DNA/RNA sequence information correlation (IC) and partial information correlation (PIC). The topology of phylogenetic tree of Betacoronavirus is remarkably consistent with biologist's systematics, classifies 2019-nCoV as Sarbecovirus of Betacoronavirus and supports the assumption that these novel viruses are of bat origin with pangolin as one of the possible intermediate hosts. The novel virus branch of phylogenetic tree shows location-virus linkage. The placement of root of the early 2019-nCoV tree is studied carefully in Neighbor Joining consensus algorithm by introducing different out-groups (Bat-related coronaviruses, Pangolin coronaviruses and HIV viruses etc.) and comparing with UPGMA consensus trees. Several oldest branches (lineages) of the 2019-nCoV tree are deduced that means the COVID-19 may begin to spread in several regions in the world before its outbreak in Wuhan.

positive: Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic There are outstanding evolutionary questions on the recent emergence of coronavirus SARS-CoV-2/hCoV-19 in Hubei province that caused the COVID-19 pandemic, including (1) the relationship of the new virus to the SARS-related coronaviruses, (2) the role of bats as a reservoir species, (3) the potential role of other mammals in the emergence event, and (4) the role of recombination in viral emergence. Here, we address these questions and find that the sarbecoviruses – the viral subgenus responsible for the emergence of SARS-CoV and SARS-CoV-2 – exhibit frequent recombination, but the SARS-CoV-2 lineage itself is not a recombinant of any viruses detected to date. In order to employ phylogenetic methods to date the divergence events between SARS-CoV-2 and the bat sarbecovirus reservoir, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Bayesian evolutionary rate and divergence date estimates were consistent for all three recombination-free alignments and robust to two different prior specifications based on HCoV-OC43 and MERS-CoV evolutionary rates. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estimated as 1948 (95% HPD: 1879-1999), 1969 (95% HPD: 1930-2000), and 1982 (95% HPD: 1948-2009). Despite intensified characterization of sarbecoviruses since SARS, the lineage giving rise to SARS-CoV-2 has been circulating unnoticed for decades in bats and been transmitted to other hosts such as pangolins. The occurrence of a third significant coronavirus emergence in 17 years together with the high prevalence and virus diversity in bats implies that these viruses are likely to cross species boundaries again. In Brief The Betacoronavirus SARS-CoV-2 is a member of the sarbecovirus subgenus which shows frequent recombination in its evolutionary history. We characterize the extent of this genetic exchange and identify non-recombining regions of the sarbecovirus genome using three independent methods to remove the effects of recombination. Using these non-recombining genome regions and prior information on coronavirus evolutionary rates, we obtain estimates from three approaches that the most likely divergence date of SARS-CoV-2 from its most closely related available bat sequences ranges from 1948 to 1982. Key Points RaTG13 is the closest available bat virus to SARS-CoV-2; a sub-lineage of these bat viruses is able to infect humans. Two sister lineages of the RaTG13/SARS-CoV-2 lineage infect Malayan pangolins. The sarbecoviruses show a pattern of deep recombination events, indicating that there are high levels of co-infection in horseshoe bats and that the viral pool can generate novel allele combinations and substantial genetic diversity; the sarbecoviruses are efficient ‘explorers’ of phenotype space. The SARS-CoV-2 lineage is not a recent recombinant, at least not involving any of the bat or pangolin viruses sampled to date. Non-recombinant regions of the sarbecoviruses can be identified, allowing for phylogenetic inference and dating to be performed. We constructed three such regions using different methods. We estimate that RaTG13 and SARS-CoV-2 diverged 40 to 70 years ago. There is a diverse unsampled reservoir of generalist viruses established in horseshoe bats. While an intermediate host responsible for the zoonotic event cannot be ruled out, the relevant evolution for spillover to humans very likely occurred in horseshoe bats.

positive: Epidemiology of SARS-CoV-2 Emergence Amidst Community-Acquired Respiratory Viruses Background. SARS-CoV-2 emerged in China in December 2019 as new cause of severe viral pneumonia (CoVID-19) reaching Europe by late January 2020. We validated the WHO-recommended assay and describe the epidemiology of SARS-CoV-2 and community-acquired respiratory viruses (CARVs). Methods. Naso-oropharyngeal swabs (NOPS) from 7663 individuals were prospectively tested by the Basel-S-gene and the WHO-based E-gene-assay (Roche) using Basel-N-gene-assay for confirmation. CARVs were tested in 2394 NOPS by multiplex-NAT, including 1816 together with SARS-CoV-2. Results. Basel-S-gene and Roche-E-gene-assays were concordant in 7475 cases (97.5%) including 825 (11%) positive samples. In 188 (2.5%) discordant cases, SARS-CoV-2 loads were significantly lower than in concordant positive ones and confirmed in 105 NOPS. Adults were more likely to test positive for SARS-CoV-2, while children were more likely to test CARV-positive. CARV co-infections with SARS-CoV-2 occurred in 1.8%. SARS-CoV-2 replaced other CARVs within 3 weeks reaching 48% of all detected respiratory viruses followed by rhino/enterovirus (13%), influenzavirus (12%), coronavirus (9%), respiratory syncytial (6%) and metapneumovirus (6%). Conclusions. The differential diagnosis for respiratory infections was broad during the early pandemic, affecting infection control and treatment decisions. We discuss the role of pre-existing immunity and competitive CARV replication for the epidemiology of SARS-CoV-2 infection among adults and children.

positive: Analysis of SARS-CoV-2 Genomes from Southern California Reveals Community Transmission Pathways in the Early Stage of the US COVID-19 Pandemic Given the higher mortality rate and widespread phenomenon of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) within the United States (US) population, understanding the mutational pattern of SARS CoV-2 has global implications for detection and therapy to prevent further escalation. Los Angeles has become an epicenter of the SARS-CoV-2 pandemic in the US. Efforts to contain the spread of SARS-CoV-2 require identifying its genetic and geographic variation and understanding the drivers of these differences. For the first time, we report genetic characterization of SARS-CoV-2 genome isolates in the Los Angeles population using targeted next generation sequencing (NGS). Samples collected at Cedars Sinai Medical Center were collected from patients with confirmed SARS-CoV-2 infection. We identified and diagnosed 192 patients by our in-house qPCR assay. In this population, the highest frequency variants were in known mutations in the 5'UTR, AA193 protein, RdRp and the spike glycoprotein. SARS-CoV-2 transmission within the local community was tracked by integrating mutation data with patient postal codes with two predominant community spread clusters being identified. Notably, significant viral genomic diversity was identified. Less than 10 percent of the Los Angeles community samples resembled published mutational profiles of SARS-CoV-2 genomes from China, while >50 percent of the isolates shared closely similarities to those from New York State. Based on these findings we conclude SARS-CoV-2 was likely introduced into the Los Angeles community predominantly from New York State but also via multiple other independent transmission routes including but not limited to Washington State and China.

positive: [Analysis of variation and evolution of SARS-CoV-2 genome]. OBJECTIVE To analyze the evolution and variation of SARS-CoV-2 during the epidemic starting at the end of 2019. METHODS We downloaded the full-length genome sequence of SARS-CoV-2 from the databases of GISAID and NCBI. Using the software for bioinformatics including MEGA-X, BEAST, and TempEst, we constructed the genomic evolution tree, inferred the time evolution signal of the virus, calculated the tMRCA time of the virus and analyzed the selection pressure of the virus during evolution. RESULTS The phylogenetic tree showed that SARS-CoV-2 belonged to the Sarbecovirus subgenus of β Coronavirus genus together with bat coronavirus BetaCoV/bat/Yunnan/RaTG13/2013, bat-SL-CoVZC45, bat-SL-CoVZXC21 and SARS-CoV. The genomic sequences of SARS-CoV-2 isolated from the ongoing epidemic showed a weak time evolution signal with an average tMRCA time of 73 days (95% CI: 38.9-119.3 days). No positive time evolution signal was found between SARS-CoV-2 and BetaCoV/bat/Yunnan/RaTG13/2013, but the former virus had a strong positive temporal evolution relationship with bat-SL-CoVZC45 and SARS-CoV. The major cause for mutations of SARS-CoV-2 was the pressure of purification selection during the epidemic. CONCLUSIONS SARS-CoV-2 may have emerged as early as November, 2019, originating most likely from bat-associated coronavirus. This finding may provide evidence for tracing the sources and evolution of the virus.

positive: Origin and Evolution of the 2019 Novel Coronavirus

positive: SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasispecies with extended ORF3b that may exacerbate COVID-19 symptoms. Highlights ORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonist SARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV ortholog The anti-IFN activity of ORF3b depends on the length of its C-terminus An ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases

positive: In silico prediction of potential inhibitors for the Main protease of SARS-CoV-2 using molecular docking and dynamics simulation based drug-repurposing BACKGROUND: The rapidly enlarging COVID-19 pandemic caused by the novel SARS-corona virus-2 is a global public health emergency of an unprecedented level. Unfortunately no treatment therapy or vaccine is yet available to counter the SARS-CoV-2 infection, which substantiates the need to expand research efforts in this direction. The indispensable function of the main protease in virus replication makes this enzyme a promising target for inhibitors screening and drug discovery to treat novel coronavirus infection. The recently concluded α-ketoamide ligand-bound X-ray crystal structure of SARS-CoV-2 M(pro) (PDB ID: 6Y2F) from Zhang et al. has revealed the potential inhibitor binding mechanism and the molecular determinants responsible for substrate binding. METHODS: For the study, we have targeted the SARS-CoV-2 M(pro) for the screening of FDA approved antiviral drugs and carried out molecular docking based virtual screening. Further molecular dynamic simulation studies of the top three selected drugs carried out to investigated for their binding affinity and stability in the SARS-CoV-2 M(pro) active site. The phylogenetic analysis was also performed to know the relatedness between the SARS-CoV-2 genomes isolated from different countries. RESULTS: The phylogenetic analysis of the SARS-CoV-2 genome reveals that the virus is closely related to the Bat-SL-CoV and does not exhibit any divergence at the genomic level. Molecular docking studies revealed that among the 77 drugs, screened top ten drugs shows good binding affinities, whereas the top three drugs: Lopinavir-Ritonavir, Tipranavir, and Raltegravir were undergone for molecular dynamics simulation studies for their conformational stability in the active site of the SARS-CoV-2 M(pro) protein. CONCLUSIONS: In the present study among the library of FDA approved antiviral drugs, the top three inhibitors Lopinavir-Ritonavir, Tipranavir, and Raltegravir show the best molecular interaction with the main protease of SARS-CoV-2. However, the in-vitro efficacy of the drug molecules screened in this study further needs to be corroborated by carrying out a biochemical and structural investigation.

positive: SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February-March 2020 We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.

positive: Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies Summary Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

positive: Genomic analysis of SARS-CoV-2 strains among Indians returning from Italy, Iran & China, & Italian tourists in India.

positive: Codon Usage and Phenotypic Divergences of SARS-CoV-2 Genes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December of 2019, causes a severe acute respiratory illness with a high mortality rate, and has spread around the world. To gain an understanding of the evolution of the newly emerging SARS-CoV-2, we herein analyzed the codon usage pattern of SARS-CoV-2. For this purpose, we compared the codon usage of SARS-CoV-2 with that of other viruses belonging to the subfamily of Orthocoronavirinae. We found that SARS-CoV-2 has a high AU content that strongly influences its codon usage, which appears to be better adapted to the human host. We also studied the evolutionary pressures that influence the codon usage of five conserved coronavirus genes encoding the viral replicase, spike, envelope, membrane and nucleocapsid proteins. We found different patterns of both mutational bias and natural selection that affect the codon usage of these genes. Moreover, we show here that the two integral membrane proteins (matrix and envelope) tend to evolve slowly by accumulating nucleotide mutations on their corresponding genes. Conversely, genes encoding nucleocapsid (N), viral replicase and spike proteins (S), although they are regarded as are important targets for the development of vaccines and antiviral drugs, tend to evolve faster in comparison to the two genes mentioned above. Overall, our results suggest that the higher divergence observed for the latter three genes could represent a significant barrier in the development of antiviral therapeutics against SARS-CoV-2.

positive: Discovery of potential multi-target-directed ligands by targeting host-specific SARS-CoV-2 structurally conserved main protease Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the current COVID-19 pandemic. Worldwide this disease has infected over 2.5 million individuals with a mortality rate ranging from 5 to 10%. There are several efforts going on in the drug discovery to control the SARS-CoV-2 viral infection. The main protease (MPro) plays a critical role in viral replication and maturation, thus can serve as the primary drug target. To understand the structural evolution of MPro, we have performed phylogenetic and Sequence Similarity Network analysis, that depicted divergence of Coronaviridae MPro in five clusters specific to viral hosts. This clustering was corroborated with the comparison of MPro structures. Furthermore, it has been observed that backbone and binding site conformations are conserved despite variation in some of the residues. These attributes can be exploited to repurpose available viral protease inhibitors against SARS-CoV-2 MPro. In agreement with this, we performed screening of â¼7100 molecules including active ingredients present in the Ayurvedic anti-tussive medicines, anti-viral phytochemicals and synthetic anti-virals against SARS-CoV-2 MPro as the primary target. We identified several natural molecules like Î´-viniferin, myricitrin, taiwanhomoflavone A, lactucopicrin 15-oxalate, nympholide A, afzelin, biorobin, hesperidin and phyllaemblicin B that strongly binds to SARS-CoV-2 MPro. Intrestingly, these molecules also showed strong binding with other potential targets of SARS-CoV-2 infection like viral receptor human angiotensin-converting enzyme 2 (hACE-2) and RNA dependent RNA polymerase (RdRp). We anticipate that our approach for identification of multi-target-directed ligand will provide new avenues for drug discovery against SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

positive: A Snapshot of SARS-CoV-2 Genome Availability up to April 2020 and its Implications: Data Analysis BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been growing exponentially, affecting over 4 million people and causing enormous distress to economies and societies worldwide. A plethora of analyses based on viral sequences has already been published both in scientific journals and through non–peer-reviewed channels to investigate the genetic heterogeneity and spatiotemporal dissemination of SARS-CoV-2. However, a systematic investigation of phylogenetic information and sampling bias in the available data is lacking. Although the number of available genome sequences of SARS-CoV-2 is growing daily and the sequences show increasing phylogenetic information, country-specific data still present severe limitations and should be interpreted with caution. OBJECTIVE: The objective of this study was to determine the quality of the currently available SARS-CoV-2 full genome data in terms of sampling bias as well as phylogenetic and temporal signals to inform and guide the scientific community. METHODS: We used maximum likelihood–based methods to assess the presence of sufficient information for robust phylogenetic and phylogeographic studies in several SARS-CoV-2 sequence alignments assembled from GISAID (Global Initiative on Sharing All Influenza Data) data released between March and April 2020. RESULTS: Although the number of high-quality full genomes is growing daily, and sequence data released in April 2020 contain sufficient phylogenetic information to allow reliable inference of phylogenetic relationships, country-specific SARS-CoV-2 data sets still present severe limitations. CONCLUSIONS: At the present time, studies assessing within-country spread or transmission clusters should be considered preliminary or hypothesis-generating at best. Hence, current reports should be interpreted with caution, and concerted efforts should continue to increase the number and quality of sequences required for robust tracing of the epidemic.

positive: Analysis of the Hosts and Transmission Paths of SARS-CoV-2 in the COVID-19 Outbreak The severe respiratory disease COVID-19 was initially reported in Wuhan, China, in December 2019, and spread into many provinces from Wuhan. The corresponding pathogen was soon identified as a novel coronavirus named SARS-CoV-2 (formerly, 2019-nCoV). As of 2 May, 2020, over 3 million COVID-19 cases had been confirmed, and 235,290 deaths had been reported globally, and the numbers are still increasing. It is important to understand the phylogenetic relationship between SARS-CoV-2 and known coronaviruses, and to identify its hosts for preventing the next round of emergency outbreak. In this study, we employ an effective alignment-free approach, the Natural Vector method, to analyze the phylogeny and classify the coronaviruses based on genomic and protein data. Our results show that SARS-CoV-2 is closely related to, but distinct from the SARS-CoV branch. By analyzing the genetic distances from the SARS-CoV-2 strain to the coronaviruses residing in animal hosts, we establish that the most possible transmission path originates from bats to pangolins to humans.

positive: Genomic Analysis of Early SARS-CoV-2 Variants Introduced in Mexico. The COVID-19 pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to implement effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, the SARS-CoV-2 lineage A/G, containing mostly sequences from North America, and the lineage B/S containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized fourteen independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represent local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described for the country. Within this local transmission cluster, we also identified the H49Y amino acid change in the Spike protein. This mutation is a homoplasy occurring independently through time and space, and may function as a molecular marker to follow on any further spread of these viral variants throughout the country. Our results depict the general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts.IMPORTANCE Understanding the introduction, spread and establishment of SARS-CoV-2 within distinct human populations is crucial to implement effective control strategies as well as the evolution of the pandemics. In this work, we describe that the initial virus strains introduced in Mexico came from Europe and the United States and the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains having the mutation H49Y in the Spike protein, that could be further used as a molecular marker to follow viral spread within the country and the region.

positive: Phylogenetic analysis of the first four SARS‐CoV‐2 cases in Chile The current pandemic caused by the new coronavirus is a worldwide public health concern. To aboard this emergency, and like never before, scientific groups around the world have been working in a fast and coordinated way to get the maximum of information about this virus when it has been almost 3 months since the first cases were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub‐classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection.

positive: Genomics of Indian SARS-CoV-2: Implications in genetic diversity, possible origin and spread of virus World Health Organization (WHO) declared COVID-19 as a pandemic disease on March 11, 2020. Comparison of genome sequences from diverse locations allows us to identify the genetic diversity among viruses which would help in ascertaining viral virulence, disease pathogenicity, origin and spread of the SARS-CoV-2 between countries. The aim of this study is to ascertain the genetic diversity among Indian SARS-CoV-2 isolates. Initial examination of the phylogenetic data of SARS-CoV-2 genomes (n=3123) from different continents deposited at GISAID (Global Initiative on Sharing All Influenza Data) revealed multiple origin for Indian isolates. An in-depth analysis of 449 viral genomes derived from samples representing countries from USA, Europe, China, East Asia, South Asia, Oceania, Middle East regions and India revealed that most Indian samples are divided into two clusters (A and B) with cluster A showing more similarity to samples from Oceania and Kuwait and the cluster B grouping with countries from Europe, Middle East and South Asia. Diversity analysis of viral clades, which are characterized by specific non-synonymous mutations in viral proteins, discovered that the cluster A Indian samples belong to I clade (V378I in ORF1ab), which is an Oceania clade with samples having Iran connections and the cluster B Indian samples belong to G clade (D614G in Spike protein), which is an European clade. Thus our study identifies that the Indian SARS-CoV-2 viruses belong to I and G clades with potential origin to be countries mainly from Oceania, Europe, Middle East and South Asia regions, which strongly implying the spread of virus through most travelled countries. The study also emphasizes the importance of pathogen genomics through phylogenetic analysis to discover viral genetic diversity and understand the viral transmission dynamics with eventual grasp on viral virulence and disease pathogenesis.

positive: Spread dynamics of SARS-CoV-2 epidemic in China: a phylogenetic analysis To reveal the detailed spread dynamics of SARS-CoV-2 epidemic in China, a phylogenetic analysis was performed by the Bayesian inference framework tool. 233 strains were retrieved from confirmed cases in China until March 31, 2020. The tMRCA of SARS-CoV-2 strains in China could be traced back to December 9, 2019. According to the effective population size curve reconstructed by Skyline model, this research revealed the influence of travel ban measures on the effective population size in China. Furthermore, we divided the epidemic process of SARS-CoV-2 in China into 4 stages according to the effective population size curve. With the Bayesian stochastic search variable selection method, phylogeographical reconstruction detailedly described the geographic spread behavior of SARS-CoV-2 in each stage and confirmed the importance of travel ban in blocking SARS-CoV-2 cross-regional spread. This article summarizes the influence of prevention and control measures in China, which has a positive impact on the world.

positive: On the evolutionary epidemiology of SARS-CoV-2 There is no doubt that the novel coronavirus SARS-CoV-2 that causes COVID-19 is mutating and thus has the potential to adapt during the current pandemic. Whether this evolution will lead to changes in the transmission, the duration, or the severity of the disease is not clear. This has led to considerable scientific and media debate, from raising alarms about evolutionary change to dismissing it. Here we review what little is currently known about the evolution of SARS-CoV-2 and extend existing evolutionary theory to consider how selection might be acting upon the virus during the COVID-19 pandemic. While there is currently no definitive evidence that SARS-CoV-2 is undergoing further adaptation, continued, evidence-based, analysis of evolutionary change is important so that public health measures can be adjusted in response to substantive changes in the infectivity or severity of COVID-19.

positive: A doubt of multiple introduction of SARS-CoV-2 in Italy: A preliminary overview The emergence of the novel betacoronavirus, recently renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised serious concerns due to the virus's rapid dissemination worldwide. Nevertheless, there is limited information about the genomic epidemiology of SARS-CoV-2 circulating in Italy from surveillance studies. The shortage of complete genomic sequences available impairs our understanding of the SARS-CoV-2 introduction and establishment in the country. To better understand its dynamics in Italy, we analyzed complete genomes of SARS-CoV-2 isolates, obtained directly from clinical samples. Our phylogenetic reconstructions suggest possible multiple introduction of SARS-CoV-2. Continued genomic surveillance strategies are needed to improve monitoring and understanding of the current SARS-CoV-2 epidemics, which might help to attenuate public health impact of infectious diseases.

positive: Virus strain from a mild COVID-19 patient in Hangzhou represents a new trend in SARS-CoV-2 evolution potentially related to Furin cleavage site The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.

positive: Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2 SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.

positive: Molecular phylogeny of coronaviruses including human SARS-CoV Phylogenetic tree of coronaviruses (CoVs) including the human SARS-associated virus is reconstructed from complete genomes by using our newly developed K-string composition approach. The relation of the human SARS-CoV to other coronaviruses, i.e. the rooting of the tree is suggested by choosing an appropriate outgroup. SARS-CoV makes a separate group closer but still distant from G2 (CoVs in mammalian host). The relation between different isolates of the human SARS virus is inferred by first constructing an ultrametric distance matrix from counting sequence variations in the genomes. The resulting tree is consistent with clinic relations between the SARS-CoV isolates. In addition to a larger variety of coronavirus genomes these results provide phylogenetic knowledge based on independent novel methodology as compared to recent phylogenetic studies on SARS-CoV.

positive: An update on the origin of SARS‐CoV‐2: Despite closest identity, bat (RaTG13) and pangolin derived coronaviruses varied in the critical binding site and O‐linked glycan residues The initial cases of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) occurred in Wuhan, China, in December 2019 and swept the world by 23 June 2020 with 8 993 659 active cases, 469 587 deaths across 216 countries, areas or territories. This strongly implies global transmission occurred before the lockdown of China. However, the initial source's transmission routes of SARS‐CoV‐2 remain obscure and controversial. Research data suggest bat (RaTG13) and pangolin carried CoV were the proximal source of SARS‐CoV‐2. In this study, we used systematic phylogenetic analysis of Coronavirinae subfamily along with wild type human SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2 strains. The key residues of the receptor‐binding domain (RBD) and O‐linked glycan were compared. SARS‐CoV‐2 strains were clustered with RaTG13 (97.41% identity), Pangolin‐CoV (92.22% identity) and Bat‐SL‐CoV (80.36% identity), forms a new clade‐2 in lineage B of beta‐CoV. The alignments of RBD contact residues to ACE2 justified? Those SARS‐CoV‐2 strains sequences were 100% identical by each other, significantly varied in RaTG13 and pangolin‐CoV. SARS‐CoV‐2 has a polybasic cleavage site with an inserted sequence of PRRA compared to RaTG13 and only PRR to pangolin. Only serine (Ser) in pangolin and both threonine (Thr) and serine (Ser) O‐linked glycans were seen in RaTG13, suggesting that a detailed study needed in pangolin (Manis javanica) and bat (Rhinolophus affinis) related CoV.

positive: Discovery of G-quadruplex-forming sequences in SARS-CoV-2 The outbreak caused by the novel coronavirus SARS-CoV-2 has been declared a global health emergency. G-quadruplex structures in genomes have long been considered essential for regulating a number of biological processes in a plethora of organisms. We have analyzed and identified 25 four contiguous GG runs (G(2)N(x)G(2)N(y)G(2)N(z)G(2)) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). Detailed analysis of SARS-CoV-2 PQSs revealed their locations in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. Identical PQSs were also found in the other members of the Coronaviridae family. The top-ranked PQSs at positions 13385 and 24268 were confirmed to form RNA G-quadruplex structures in vitro by multiple spectroscopic assays. Furthermore, their direct interactions with viral helicase (nsp13) were determined by microscale thermophoresis. Molecular docking model suggests that nsp13 distorts the G-quadruplex structure by allowing the guanine bases to be flipped away from the guanine quartet planes. Targeting viral helicase and G-quadruplex structure represents an attractive approach for potentially inhibiting the SARS-CoV-2 virus.

positive: Laboratory based surveillance of SARS-CoV-2 in Pakistan COVID-19 cases are alarmingly increasing in Pakistan since May 2020. Laboratory based surveillance system has been in place since the start of the pandemic. The genomic surveillance of SARS-CoV-2 strains isolated locally has been conducted based on partial ORF1b. The sequences were classified to show the phylogenetic correlation and showed 100% homology with those detected in neighboring countries India and China. The rapid increase in cases has led to development of robust strategies to enhance the laboratory testing capacity. We are currently meeting the country requirement to diagnose the virus in the community. Nonetheless, factors like recent ease in lockdown measures has led to massive rise in number of cases in few weeks time.

positive: An emergent clade of SARS-CoV-2 linked to returned travellers from Iran The SARS-CoV-2 epidemic has rapidly spread outside China with major outbreaks occurring in Italy, South Korea and Iran. Phylogenetic analyses of whole genome sequencing data identified a distinct SARS-CoV-2 clade linked to travellers returning from Iran to Australia and New Zealand. This study highlights potential viral diversity driving the epidemic in Iran, and underscores the power of rapid genome sequencing and public data sharing to improve the detection and management of emerging infectious diseases.

positive: Detailed phylogenetic analysis of SARS-CoV-2 reveals latent capacity to bind human ACE2 receptor SARS-CoV-2 is a once-in-a-century pandemic, having emerged suddenly as a highly infectious viral pathogen. Previous phylogenetic analyses show its closest known evolutionary relative to be a virus isolated from bats (RaTG13), with a common assumption that SARS-CoV-2 evolved from a zoonotic ancestor via recent genetic changes (likely in the Spike protein receptor binding domain – or RBD) that enabled it to infect humans. We used detailed phylogenetic analysis, ancestral sequence reconstruction, and molecular dynamics simulations to examine the Spike-RBD’s functional evolution, finding to our surprise that it has likely possessed high affinity for human cell targets since at least 2013.

positive: CoronaHiT: large scale multiplexing of SARS-CoV-2 genomes using Nanopore sequencing The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 24 samples at a time. There is a need for a higher throughput method to reduce cost per genome. Here we present CoronaHiT, a method capable of multiplexing up to 95 small genomes on a single Nanopore flowcell, which uses transposase mediated addition of adapters and PCR based addition of symmetric barcodes. We demonstrate the method using 48 and 94 SARS-CoV-2 genomes per flowcell, amplified using the ARTIC protocol, and compare performance with Illumina and ARTIC ONT sequencing. Results demonstrate that all sequencing methods produce inaccurate genomes when the RNA extract from SARS-CoV-2 positive clinical sample has a cycle threshold (Ct) >= 32. Results from set same set of 23 samples with a broad range of Cts show that the consensus genomes have >90% coverage (GISAID criteria) for 78.2% of samples for CoronaHiT-48, 73.9% for CoronaHiT-94, 78.2% for Illumina and 73.9% for ARTIC ONT, and all have the same clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 94 SARS-CoV-2 genomes per nanopore flowcell without compromising the quality of the resulting genomes while reducing library preparation complexity and significantly reducing cost. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

positive: A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster BACKGROUND: An ongoing outbreak of pneumonia associated with a novel coronavirus was reported in Wuhan city, Hubei province, China. Affected patients were geographically linked with a local wet market as a potential source. No data on person-to-person or nosocomial transmission have been published to date. METHODS: In this study, we report the epidemiological, clinical, laboratory, radiological, and microbiological findings of five patients in a family cluster who presented with unexplained pneumonia after returning to Shenzhen, Guangdong province, China, after a visit to Wuhan, and an additional family member who did not travel to Wuhan. Phylogenetic analysis of genetic sequences from these patients were done. FINDINGS: From Jan 10, 2020, we enrolled a family of six patients who travelled to Wuhan from Shenzhen between Dec 29, 2019 and Jan 4, 2020. Of six family members who travelled to Wuhan, five were identified as infected with the novel coronavirus. Additionally, one family member, who did not travel to Wuhan, became infected with the virus after several days of contact with four of the family members. None of the family members had contacts with Wuhan markets or animals, although two had visited a Wuhan hospital. Five family members (aged 36–66 years) presented with fever, upper or lower respiratory tract symptoms, or diarrhoea, or a combination of these 3–6 days after exposure. They presented to our hospital (The University of Hong Kong-Shenzhen Hospital, Shenzhen) 6–10 days after symptom onset. They and one asymptomatic child (aged 10 years) had radiological ground-glass lung opacities. Older patients (aged >60 years) had more systemic symptoms, extensive radiological ground-glass lung changes, lymphopenia, thrombocytopenia, and increased C-reactive protein and lactate dehydrogenase levels. The nasopharyngeal or throat swabs of these six patients were negative for known respiratory microbes by point-of-care multiplex RT-PCR, but five patients (four adults and the child) were RT-PCR positive for genes encoding the internal RNA-dependent RNA polymerase and surface Spike protein of this novel coronavirus, which were confirmed by Sanger sequencing. Phylogenetic analysis of these five patients' RT-PCR amplicons and two full genomes by next-generation sequencing showed that this is a novel coronavirus, which is closest to the bat severe acute respiatory syndrome (SARS)-related coronaviruses found in Chinese horseshoe bats. INTERPRETATION: Our findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings, and the reports of infected travellers in other geographical regions. FUNDING: The Shaw Foundation Hong Kong, Michael Seak-Kan Tong, Respiratory Viral Research Foundation Limited, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, Sanming Project of Medicine (Shenzhen), and High Level-Hospital Program (Guangdong Health Commission).

positive: Potential T-cell and B-cell Epitopes of 2019-nCoV As of early March, 2019-nCoV has infected more than one hundred thousand people and claimed thousands of lives. 2019-nCoV is a novel form of coronavirus that causes COVID-19 and has high similarity with SARS-CoV. No approved vaccine yet exists for any form of coronavirus. Here we use computational tools from structural biology and machine learning to identify 2019-nCoV T-cell and B-cell epitopes based on viral protein antigen presentation and antibody binding properties. These epitopes can be used to develop more effective vaccines and identify neutralizing antibodies. We identified 405 viral peptides with good antigen presentation scores for both human MHC-I and MHC-II alleles, and two potential neutralizing B-cell epitopes near the 2019-nCoV spike protein receptor binding domain (440-460 and 494-506). Analyzing mutation profiles of 68 viral genomes from four continents, we identified 96 coding-change mutations. These mutations are more likely to occur in regions with good MHC-I presentation scores (p=0.02). No mutations are present near the spike protein receptor binding domain. Based on these findings, the spike protein is likely immunogenic and a potential vaccine candidate. We validated our computational pipeline with SARS-CoV experimental data. The novel coronavirus 2019-nCoV has affected more than 100 countries and continues to spread. There is an immediate need for effective vaccines that contain antigens which trigger responses from human T-cells and B-cells (known as epitopes). Here we identify potential T-cell epitopes through an analysis of human antigen presentation, as well as B-cell epitopes through an analysis of protein structure. We identify a list of top candidates, including an epitope located on 2019-nCoV spike protein that potentially triggers both T-cell and B-cell responses. Analyzing 68 samples, we observe that viral mutations are more likely to happen in regions with strong antigen presentation, a potential form of immune evasion. Our computational pipeline is validated with experimental data from SARS-CoV.

positive: On the origin and continuing evolution of SARS-CoV-2 The SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China, and has since impacted a large portion of China and raised major global concern. Herein, we investigated the extent of molecular divergence between SARS-CoV-2 and other related coronaviruses. Although we found only 4% variability in genomic nucleotides between SARS-CoV-2 and a bat SARS-related coronavirus (SARSr-CoV; RaTG13), the difference at neutral sites was 17%, suggesting the divergence between the two viruses is much larger than previously estimated. Our results suggest that the development of new variations in functional sites in the receptor-binding domain (RBD) of the spike seen in SARS-CoV-2 and viruses from pangolin SARSr-CoVs are likely caused by mutations and natural selection besides recombination. Population genetic analyses of 103 SARS-CoV-2 genomes indicated that these viruses evolved into two major types (designated L and S), that are well defined by two different SNPs that show nearly complete linkage across the viral strains sequenced to date. Although the L type (∼70%) is more prevalent than the S type (∼30%), the S type was found to be the ancestral version. Whereas the L type was more prevalent in the early stages of the outbreak in Wuhan, the frequency of the L type decreased after early January 2020. Human intervention may have placed more severe selective pressure on the L type, which might be more aggressive and spread more quickly. On the other hand, the S type, which is evolutionarily older and less aggressive, might have increased in relative frequency due to relatively weaker selective pressure. These findings strongly support an urgent need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with coronavirus disease 2019 (COVID-19).

positive: Tracking the COVID-19 pandemic in Australia using genomics BACKGROUND: Whole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODS: Cases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTS: Between 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 samples were included in genomic analyses. Sequenced samples from Australia were representative of the global diversity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified; these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential samples from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing. CONCLUSIONS: Our data provide a comprehensive framework for the use of SARS-CoV-2 genomics in public health responses. The application of genomics to rapidly identify SARS-CoV-2 transmission chains will become critically important as social restrictions ease globally. Public health responses to emergent cases must be swift, highly focused and effective.

positive: Genetic variants and source of introduction of SARS‐CoV‐2 in South America After more than four months of the COVID‐19 pandemics with genomic information of SARS‐CoV‐2 around the globe, there are more than 1000 complete genomes of this virus. We used 691 genomes from the GISAID database. Several studies have been reporting mutations and hotspots according to the viral evolution. Our work intends to show and compare positions that have variants in 30 complete viral genomes from South American countries. We classified strains according to point alterations and portray the source where strains came into this region. Most viruses entered to South America from Europe, followed by Oceania. Only Chilean isolates demonstrated a relationship to Asian isolates. Some changes in South American genomes are near to specific domains related to replication or S protein. Our work contributes to global understanding of which sort of strains are spreading throughout South American countries, and the differences among them according to the first isolates introduced in this region. This article is protected by copyright. All rights reserved.

positive: SARS-CoV-2 Molecular and Phylogenetic analysis in COVID-19 patients: A preliminary report from Iran BACKGROUND: The aim of the current study was to investigate and track the SARS-CoV-2 in Iranian Coronavirus Disease 2019 (COVID-19) patients using molecular and phylogenetic methods. METHODS: We enrolled seven confirmed cases of COVID-19 patients for the phylogenetic assessment of the SARS-CoV-2 in Iran. The nsp-2, nsp-12, and S genes were amplified using one-step RT-PCR and sequenced using Sanger sequencing method. Popular bioinformatics software were used for sequences alignment and analysis as well as phylogenetic construction. RESULTS: The mean age of the patients in the present study was 60.42 ± 9.94 years and 57.1% (4/7) were male. The results indicated high similarity between Iranian and Chinese strains. We could not find any particular polymorphisms in the assessed regions of the three genes. Phylogenetic trees by neighbor-joining and maximum likelihood method of nsp-2, nsp-12, and S genes showed that there are not any differences between Iranian isolates and those of other countries. CONCLUSION: As a preliminary phylogenetic study in Iranian SARS-CoV-2 isolates, we found that these isolates are closely related to the Chinese and reference sequences. Also, no sensible differences were observed between Iranian isolates and those of other countries. Further investigations are recommended using more comprehensive methods and larger sample sizes.

positive: Molecular Evolution of SARS-CoV-2 Structural Genes: Evidence of Positive Selection in Spike Glycoprotein SARS-CoV-2 caused a global pandemic in early 2020 and has resulted in more than 8,000,000 infections as well as 430,000 deaths in the world so far. Four structural proteins, envelope (E), membrane (M), nucleocapsid (N) and spike (S) glycoprotein, play a key role in controlling the entry into human cells and virion assembly of SARS-CoV-2. However, how these genes evolve during its human to human transmission is largely unknown. In this study, we screened and analyzed roughly 3090 SARS-CoV-2 isolates from GenBank database. The distribution of the four gene alleles is determined:16 for E, 40 for M, 131 for N and 173 for S genes. Phylogenetic analysis shows that global SARS-CoV-2 isolates can be clustered into three to four major clades based on the protein sequences of these genes. Intragenic recombination event isn’t detected among different alleles. However, purifying selection has conducted on the evolution of these genes. By analyzing full genomic sequences of these alleles using codon-substitution models (M8, M3 and M2a) and likelihood ratio tests (LRTs) of codeML package, it reveals that codon 614 of S glycoprotein has subjected to strong positive selection pressure and a persistent D614G mutation is identified. The definitive positive selection of D614G mutation is further confirmed by internal fixed effects likelihood (IFEL) and Evolutionary Fingerprinting methods implemented in Hyphy package. In addition, another potential positive selection site at codon 5 in the signal sequence of the S protein is also identified. The allele containing D614G mutation has undergone significant expansion during SARS-CoV-2 global pandemic, implying a better adaptability of isolates with the mutation. However, L5F allele expansion is relatively restricted. The D614G mutation is located at the subdomain 2 (SD2) of C-terminal portion (CTP) of the S1 subunit. Protein structural modeling shows that the D614G mutation may cause the disruption of salt bridge among S protein monomers increase their flexibility, and in turn promote receptor binding domain (RBD) opening, virus attachment and entry into host cells. Located at the signal sequence of S protein as it is, L5F mutation may facilitate the protein folding, assembly, and secretion of the virus. This is the first evidence of positive Darwinian selection in the spike gene of SARS-CoV-2, which contributes to a better understanding of the adaptive mechanism of this virus and help to provide insights for developing novel therapeutic approaches as well as effective vaccines by targeting on mutation sites.

positive: COVID-19: a new challenge for human beings

positive: Genomic epidemiology of SARS-CoV-2 in Colombia Coronavirus disease 2019 (COVID-19) was first diagnosed in Colombia from a traveler arriving from Italy on February 26, 2020. To date, available data on the origins and number or introductions of SARS-CoV-2 into the country are limited. Here, we sequenced SARS-CoV-2 from 43 clinical samples and-together with another 73 genomes sequences available from the country-we investigated the emergence and the routes of importation of COVID-19 into Colombia using epidemiological, historical air travel and phylogenetic observations. Our study provided evidence of multiple introductions, mostly from Europe, with at least 12 lineages being documented. Phylogenetic findings validated the linkage of epidemiologically-linked transmission chains. Our results demonstrate the advantages of genome sequencing to complement COVID-19 outbreak investigation and underscores that human mobility and genetic data are relevant to complete epidemiological investigation and better characterize virus transmission dynamics at local scales.

positive: Mutations, Recombination and Insertion in the Evolution of 2019-nCoV The 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) has spread more rapidly than any other betacoronavirus including SARS-CoV and MERS-CoV. However, the mechanisms responsible for infection and molecular evolution of this virus remained unclear. We collected and analyzed 120 genomic sequences of 2019-nCoV including 11 novel genomes from patients in China. Through comprehensive analysis of the available genome sequences of 2019-nCoV strains, we have tracked multiple inheritable SNPs and determined the evolution of 2019-nCoV relative to other coronaviruses. Systematic analysis of 120 genomic sequences of 2019-nCoV revealed co-circulation of two genetic subgroups with distinct SNPs markers, which can be used to trace the 2019-nCoV spreading pathways to different regions and countries. Although 2019-nCoV, human and bat SARS-CoV share high homologous in overall genome structures, they evolved into two distinct groups with different receptor entry specificities through potential recombination in the receptor binding regions. In addition, 2019-nCoV has a unique four amino acid insertion between S1 and S2 domains of the spike protein, which created a potential furin or TMPRSS2 cleavage site. Our studies provided comprehensive insights into the evolution and spread of the 2019-nCoV. Our results provided evidence suggesting that 2019-nCoV may increase its infectivity through the receptor binding domain recombination and a cleavage site insertion. Novel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.

positive: Genomic epidemiology of SARS-CoV-2 in Guangdong Province, China Highlights: 1) 1.6 million molecular diagnostic tests identified 1,388 SARS-CoV-2 infections in Guangdong Province, China, by 19th March 2020; 2) Virus genomes can be recovered using a variety of sequencing approaches from a range of patient samples. 3) Genomic analyses reveal multiple virus importations into Guangdong Province, resulting in genetically distinct clusters that require careful interpretation. 4) Large-scale epidemiological surveillance and intervention measures were effective in interrupting community transmission in Guangdong Summary: COVID-19 is caused by the SARS-CoV-2 coronavirus and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain due to low virus genetic variation early in the pandemic. Our results illustrate how the timing, size and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required as the number of cases imported from other countries is increasing.

positive: Rampant C→U Hypermutation in the Genomes of SARS-CoV-2 and Other Coronaviruses: Causes and Consequences for Their Short- and Long-Term Evolutionary Trajectories The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has motivated an intensive analysis of its molecular epidemiology following its worldwide spread. To understand the early evolutionary events following its emergence, a data set of 985 complete SARS-CoV-2 sequences was assembled. Variants showed a mean of 5.5 to 9.5 nucleotide differences from each other, consistent with a midrange coronavirus substitution rate of 3 × 10(−4) substitutions/site/year. Almost one-half of sequence changes were C→U transitions, with an 8-fold base frequency normalized directional asymmetry between C→U and U→C substitutions. Elevated ratios were observed in other recently emerged coronaviruses (SARS-CoV, Middle East respiratory syndrome [MERS]-CoV), and decreasing ratios were observed in other human coronaviruses (HCoV-NL63, -OC43, -229E, and -HKU1) proportionate to their increasing divergence. C→U transitions underpinned almost one-half of the amino acid differences between SARS-CoV-2 variants and occurred preferentially in both 5′ U/A and 3′ U/A flanking sequence contexts comparable to favored motifs of human APOBEC3 proteins. Marked base asymmetries observed in nonpandemic human coronaviruses (U ≫ A > G ≫ C) and low G+C contents may represent long-term effects of prolonged C→U hypermutation in their hosts. The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short- and long-term evolution. Repeated cycles of mutation and reversion in favored mutational hot spots and the widespread occurrence of amino acid changes with no adaptive value for the virus represent a quite different paradigm of virus sequence change from neutral and Darwinian evolutionary frameworks and are not incorporated by standard models used in molecular epidemiology investigations. IMPORTANCE The wealth of accurately curated sequence data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its long genome, and its low substitution rate provides a relatively blank canvas with which to investigate effects of mutational and editing processes imposed by the host cell. The finding that a large proportion of sequence change in SARS-CoV-2 in the initial months of the pandemic comprised C→U mutations in a host APOBEC-like context provides evidence for a potent host-driven antiviral editing mechanism against coronaviruses more often associated with antiretroviral defense. In evolutionary terms, the contribution of biased, convergent, and context-dependent mutations to sequence change in SARS-CoV-2 is substantial, and these processes are not incorporated by standard models used in molecular epidemiology investigations.

positive: On spatial molecular arrangements of SARS-CoV2 genomes of Indian patients A pandemic caused by the SARS-CoV2 is being experienced by the whole world since December, 2019. A thorough understanding beyond just sequential similarities among the protein coding genes of SARS-CoV2 is important in order to differentiate or relate to the other known CoVs of the same genus. In this study, we compare three genomes namely MT012098 (India-Kerala), MT050493 (India-Kerala), MT358637 (India-Gujrat) from India with NC_045512 (China-Wuhan) to view the spatial as well as molecular arrangements of nucleotide bases of all the genes embedded in these four genomes. Based on different features extracted for each gene embedded in these genomes, corresponding phylogenetic relationships have been built up. Differences in phylogenetic tree arrangement with individual gene suggest that three genomes of Indian origin have come from three different origins or the evolution of viral genome is very fast process. This study would also help to understand the virulence factors, disease pathogenicity, origin and transmission of the SARS-CoV2.

positive: Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event Background A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. Methods Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. Results Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat-SARS-like coronavirus sequences. Specifically, in the 5’-part spanning the first 11,498 nucleotides and the last 3’-part spanning 24,341-30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat-SARS-like coronavirus sequences, whereas in the middle region spanning the 3’-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. Conclusions The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

positive: Pangolins Harbor SARS-CoV-2-Related Coronaviruses The pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has posed a severe threat to global public health. Yet, the origin of SARS-CoV-2 remains mysterious. Several recent studies (e.g., Lam et al ., Xiao et al .) identified SARS-CoV-2-related viruses in pangolins, providing novel insights into the evolution and diversity of SARS-CoV-2-related viruses.

positive: Coding-Complete Genome Sequence of SARS-CoV-2 Isolate from Bangladesh by Sanger Sequencing. A coding-complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate was revealed. The sample for the virus was isolated from a female patient from Dhaka, Bangladesh, suffering from coronavirus disease-2019 (COVID-19).

positive: Novel coronavirus takes flight from bats?

positive: Exploring the coronavirus epidemic using the new WashU Virus Genome Browser Since its debut in mid-December, 2019, the novel coronavirus (2019-nCoV) has rapidly spread from its origin in Wuhan, China, to several countries across the globe, leading to a global health crisis. As of February 7, 2020, 44 strains of the virus have been sequenced and uploaded to NCBI’s GenBank [1], providing insight into the virus’s evolutionary history and pathogenesis. Here, we present the WashU Virus Genome Browser, a web-based portal for viewing virus genomic data. The browser is home to 16 complete 2019-nCoV genome sequences, together with hundreds of related viral sequences including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Ebola virus. In addition, the browser features unique customizability, supporting user-provided upload of novel viral sequences in various formats. Sequences can be viewed in both a track-based representation as well as a phylogenetic tree-based view, allowing the user to easily compare sequence features across multiple strains. The WashU Virus Genome Browser inherited many features and track types from the WashU Epigenome Browser, and additionally incorporated a new type of SNV track to address the specific needs of viral research. Our Virus Browser portal can be accessed at https://virusgateway.wustl.edu, and documentation is available at https://virusgateway.readthedocs.io/.

positive: Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: Similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism The spread of COVID-19 caused by the SARS-CoV-2 outbreak has been growing since its first identification in December 2019. The publishing of the first SARS-CoV-2 genome made a valuable source of data to study the details about its phylogeny, evolution, and interaction with the host. Protein-protein binding assays have confirmed that Angiotensin-converting enzyme 2 (ACE2) is more likely to be the cell receptor through which the virus invades the host cell. In the present work, we provide an insight into the interaction of the viral spike Receptor Binding Domain (RBD) from different coronavirus isolates with host ACE2 protein. By calculating the binding energy score between RBD and ACE2, we highlighted the putative jump in the affinity from a progenitor form of SARS-CoV-2 to the current virus responsible for COVID-19 outbreak. Our result was consistent with previously reported phylogenetic analysis and corroborates the opinion that the interface segment of the spike protein RBD might be acquired by SARS-CoV-2 via a complex evolutionary process rather than a progressive accumulation of mutations. We also highlighted the relevance of Q493 and P499 amino acid residues of SARS-CoV-2 RBD for binding to human ACE2 and maintaining the stability of the interface. Moreover, we show from the structural analysis that it is unlikely for the interface residues to be the result of genetic engineering. Finally, we studied the impact of eight different variants located at the interaction surface of ACE2, on the complex formation with SARS-CoV-2 RBD. We found that none of them is likely to disrupt the interaction with the viral RBD of SARS-CoV-2.

positive: Intra-genome variability in the dinucleotide composition of SARS-CoV-2 CpG dinucleotides are under-represented in the genomes of single stranded RNA viruses, and coronaviruses, including SARS-CoV-2, are no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development, and if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the newly emerged SARS-CoV-2 genome was characterised in the context of other coronaviruses. CpG suppression amongst coronaviruses does not significantly differ according to genera of virus, but does vary according to host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. While ORF10 is only present in the genomes of a subset of coronaviruses, E is essential for virus replication. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. Phylogeny indicates that this is an ancestrally-derived trait reflecting their origin in bats, rather than something selected for after zoonotic transfer. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.

positive: A dynamic nomenclature proposal for SARS-CoV-2 to assist genomic epidemiology The ongoing pandemic spread of a novel human coronavirus, SARS-COV-2, associated with severe pneumonia disease (COVID-19), has resulted in the generation of thousands of virus genome sequences. The rate of genome generation is unprecedented, yet there is currently no coherent nor accepted scheme for naming the expanding phylogenetic diversity of SARS-CoV-2. We present a rational and dynamic virus nomenclature that uses a phylogenetic framework to identify those lineages that contribute most to active spread. Our system is made tractable by constraining the number and depth of hierarchical lineage labels and by flagging and declassifying virus lineages that become unobserved and hence are likely inactive. By focusing on active virus lineages and those spreading to new locations this nomenclature will assist in tracking and understanding the patterns and determinants of the global spread of SARS-CoV-2.

positive: Analysis of codon usage and evolutionary rates of the 2019-nCoV genes Severe acute respiratory syndrome coronavirus 2 (2019-nCoV), which first broke out in Wuhan (China) in December of 2019, causes a severe acute respiratory illness with a mortality ranging from 3% to 6%. To better understand the evolution of the newly emerging 2019-nCoV, in this paper, we analyze the codon usage pattern of 2019-nCoV. For this purpose, we compare the codon usage of 2019-nCoV with that of other 30 viruses belonging to the subfamily of orthocoronavirinae. We found that 2019-nCoV shows a rich composition of AT nucleotides that strongly influences its codon usage, which appears to be not optimized to human host. Then, we study the evolutionary pressures influencing the codon usage and evolutionary rates of the sequences of five conserved genes that encode the corresponding proteins (viral replicase, spike, envelope, membrane and nucleocapsid) characteristic of coronaviruses. We found different patterns of both mutational bias and nature selection that affect the codon usage of these genes at different extents. Moreover, we show that the two integral membrane proteins proteins (matrix and envelope) tend to evolve slowly by accumulating nucleotide mutations on their genes. Conversely, genes encoding nucleocapsid (N), viral replicase and spike proteins are important targets for the development of vaccines and antiviral drugs, tend to evolve faster as compared to other ones. Taken together, our results suggest that the higher evolutionary rate observed for these two genes could represent a major barrier in the development of antiviral therapeutics 2019-nCoV.

positive: Genomic analysis of early SARS-CoV-2 strains introduced in Mexico The COVID-19 pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to implement effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, the SARS-CoV-2 lineage A/G, containing mostly sequences from North America, and the lineage B/S containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized fourteen independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represent local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described in the country. Within this Mexican cluster, we also identified an H49Y amino acid change in the spike protein. This mutation is a homoplasy occurring independently through time and space, and may function as a molecular marker to follow on any further spread of these viral variants throughout the country. Our results depict the general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts. This work is the result of the collaboration of five institutions into one research consortium: three public health institutes and two universities. From the beginning of this work, it was agreed that the experimental leader of each institution would share the first authorship. Those were the criteria followed to assign first co-first authorship in this manuscript. The order of the other authors was randomly assigned. IMPORTANCE Understanding the introduction, spread and establishment of SARS-CoV-2 within distinct human populations is crucial to implement effective control strategies as well as the evolution of the pandemics. In this work, we describe that the initial virus strains introduced in Mexico came from Europe and the United States and the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains having the mutation H49Y in the Spike protein, that could be further used as a molecular marker to follow viral spread within the country and the region.

positive: Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality? The increasing number of deaths due to the COVID-19 pandemic has raised serious global concerns. Increased testing capacity and ample intensive care availability could explain lower mortality in some countries compared to others. Nevertheless, it is also plausible that the SARS-CoV-2 mutations giving rise to different phylogenetic clades are responsible for the apparent death rate disparities around the world. Current research literature linking the genetic make-up of SARS-CoV-2 with fatalities is lacking. Here, we suggest that this disparity in fatality rates may be attributed to SARS-CoV-2 evolving mutations and urge the international community to begin addressing the phylogenetic clade classification of SARS-CoV-2 in relation to clinical outcomes.

positive: Zoonotic origins of human coronaviruses Mutation and adaptation have driven the co-evolution of coronaviruses (CoVs) and their hosts, including human beings, for thousands of years. Before 2003, two human CoVs (HCoVs) were known to cause mild illness, such as common cold. The outbreaks of severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) have flipped the coin to reveal how devastating and life-threatening an HCoV infection could be. The emergence of SARS-CoV-2 in central China at the end of 2019 has thrusted CoVs into the spotlight again and surprised us with its high transmissibility but reduced pathogenicity compared to its sister SARS-CoV. HCoV infection is a zoonosis and understanding the zoonotic origins of HCoVs would serve us well. Most HCoVs originated from bats where they are non-pathogenic. The intermediate reservoir hosts of some HCoVs are also known. Identifying the animal hosts has direct implications in the prevention of human diseases. Investigating CoV-host interactions in animals might also derive important insight on CoV pathogenesis in humans. In this review, we present an overview of the existing knowledge about the seven HCoVs, with a focus on the history of their discovery as well as their zoonotic origins and interspecies transmission. Importantly, we compare and contrast the different HCoVs from a perspective of virus evolution and genome recombination. The current CoV disease 2019 (COVID-19) epidemic is discussed in this context. In addition, the requirements for successful host switches and the implications of virus evolution on disease severity are also highlighted.

positive: Genomic analysis and comparative multiple sequences of SARS-CoV2 BACKGROUND: China announced an outbreak of new coronavirus in the city of Wuhan on December 31, 2019; lash to now, the virus transmission has become pandemic worldwide. Severe cases from the Huanan Seafood Wholesale market in Wuhan were confirmed pneumonia with a novel coronavirus (2019-nCoV). Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies. Thus, we defined the correlation in 10 severe acute respiratory syndrome coronavirus (SARS-CoV2) sequences from different countries to analyze the genomic patterns of disease origin and evolution aiming for developing new control pandemic processes. METHODS: We apply genomic analysis to observe SARS-CoV2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1), MN985325 (USA: WA, UW), MN996527 (China, C2), MT007544 (Australia: Victoria, A1), MT027064 (USA: CA, UC), MT039890 (South Korea, K1), MT066175 (Taiwan, T1), MT066176 (Taiwan, T2), LC528232 (Japan, J1), and LC528233 (Japan, J2) for genomic sequence alignment analysis. Multiple Sequence Alignment by Clustalw (https://www.genome.jp/tools-bin/clustalw) web service is applied as our alignment tool. RESULTS: We analyzed 10 sequences from the National Center for Biotechnology Information (NCBI) database by genome alignment and found no difference in amino acid sequences within M and N proteins. There are two amino acid variances in the spike (S) protein region. One mutation found from the South Korea sequence is verified. Two possible "L" and "S" SNPs found in ORF1ab and ORF8 regions are detected. CONCLUSION: We performed genomic analysis and comparative multiple sequences of SARS-CoV2. Studies about the biological symptoms of SARS-CoV2 in clinic animals and humans will manipulate an understanding on the origin of pandemic crisis.

positive: Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India Background & objectives: Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019. Methods: Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp. Results: Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1. Interpretation & conclusions: This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

positive: Automatic Identification of SARS Coronavirus using Compression-Complexity Measures Finding vaccine or specific antiviral treatment for global pandemic of virus diseases (such as the ongoing COVID-19) requires rapid analysis, annotation and evaluation of metagenomic libraries to enable a quick and efficient screening of nucleotide sequences. Traditional sequence alignment methods are not suitable and there is a need for fast alignment-free techniques for sequence analysis. Information theory and data compression algorithms provide a rich set of mathematical and computational tools to capture essential patterns in biological sequences. In 2013, our research group (Nagaraj et al., Eur. Phys. J. Special Topics 222(3-4), 2013) has proposed a novel measure known as Effort-To-Compress (ETC) based on the notion of compression-complexity to capture the information content of sequences. In this study, we propose a compression-complexity based distance measure for automatic identification of SARS coronavirus strains from a set of viruses using only short fragments of nucleotide sequences. We also demonstrate that our proposed method can correctly distinguish SARS-CoV-2 from SARS-CoV-1 viruses by analyzing very short segments of nucleotide sequences. This work could be extended further to enable medical practitioners in automatically identifying and characterizing SARS coronavirus strain in a fast and efficient fashion using short and/or incomplete segments of nucleotide sequences. Potentially, the need for sequence assembly can be circumvented. Note The main ideas and results of this research were first presented at the International Conference on Nonlinear Systems and Dynamics (CNSD-2013) held at Indian Institute of Technology, Indore, December 12, 2013. In this manuscript, we have extended our preliminary analysis to include SARS-CoV-2 virus as well.

positive: Response to Carletti et al, "About the origin of the first two SARS-CoV-2 infections in Italy: Inference not supported by appropriate sequence analysis"

positive: Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic The COVID-19 pandemic spread very fast around the world. A few days after the first detected case in South Africa, an infection started a large hospital outbreak in Durban, KwaZulu-Natal. Phylogenetic analysis of SARS-CoV-2 genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. In this manuscript, we outline the obstacles we encountered in order to genotype SARS-CoV-2 in real-time during an urgent outbreak investigation. In this process, we encountered problems with the length of the original genotyping protocol, reagent stockout and sample degradation and storage. However, we managed to set up three different library preparation methods for sequencing in Illumina. We also managed to decrease the hands on library preparation time from twelve to three hours, which allowed us to complete the outbreak investigation in just a few weeks. We also fine-tuned a simple bioinformatics workflow for the assembly of high-quality genomes in real-time. In order to allow other laboratories to learn from our experience, we released all of the library preparation and bioinformatics protocols publicly and distributed them to other laboratories of the South African Network for Genomics Surveillance (SANGS) consortium.

positive: Genome-wide analysis of Indian SARS-CoV-2 genomes for the identification of genetic mutation and SNP The wave of COVID-19 is a big threat to the human population. Presently, the world is going through different phases of lock down in order to stop this wave of pandemic; India being no exception. We have also started the lock down on 23rd March 2020. In this current situation, apart from social distancing only a vaccine can be the proper solution to serve the population of human being. Thus it is important for all the nations to perform the genome-wide analysis in order to identify the genetic variation in Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) so that proper vaccine can be designed. This fast motivated us to analyze publicly available 566 Indian complete or near complete SARS-CoV-2 genomes to find the mutation points as substitution, deletion and insertion. In this regard, we have performed the multiple sequence alignment in presence of reference sequence from NCBI. After the alignment, a consensus sequence is build to analyze each genome in order to identify the mutation points. As a consequence, we have found 933 substitutions, 2449 deletions and 2 insertions, in total 3384 unique mutation points, in 566 genomes across 29.9 K bp. Further, it has been classified into three groups as 100 clusters of mutations (mostly deletions), 1609 point mutations as substitution, deletion and insertion and 64 SNPs. These outcomes are visualized using BioCircos and bar plots as well as plotting entropy value of each genomic location. Moreover, phylogenetic analysis has also been performed to see the evolution of SARS-CoV-2 virus in India. It also shows the wide variation in tree which indeed vivid in genomic analysis. Finally, these SNPs can be the useful target for virus classification, designing and defining the effective dose of vaccine for the heterogeneous population.

positive: Phylogenomic proximity and comparative proteomic analysis of SARS-CoV-2 The coronavirus disease (COVID-19) belongs to the family Severe Acute Respiratory Syndrome (SARS-CoV). It can be more severe for some persons and can lead to pneumonia or breathing difficulties resulting in the death of immune-compromised patients. We performed a phylogenomic and phylogeographic tree from the collected datasets. Phylogenomic analysis or sequence-based phylogeny showed an evolutionary relationship between the geographical strains. The phylogenomic tree grouped into two major clades consists of various isolates of SARS-CoV-2 and Bat SARS-like coronavirus, Bat coronavirus, and Pangolin coronavirus. The phylogenetic neighbor of newly sequenced Indian strains (Accession: MT012098.1, MT050493.1) was revealed to identify the variations between the nCoV-19 strains. The results showed keen evidence that SARS-CoV-2 has evolved from Bat SARS-like coronavirus. The evolutionary history and comparative proteomic analysis provide a new avenue for the current scientific research related to the coronavirus.

positive: Comprehensive variant and haplotype landscapes of 50,500 global SARS-CoV-2 isolates and accelerating accumulation of country-private variant profiles Understanding the genetic etiology of COVID-19 requires a comprehensive understanding of the variant and haplotype landscape of all reported genomes of SARS-COV-2, the causative virus of the disease. Country-, state/region- and possibly even city-private variant profiles may contribute to varied disease exemplifications and fatality rates observed across the globe along with host factors such as age, ethnicity and comorbidity. The Children’s Hospital of Los Angeles (CHLA) COVID-19 Analysis Research Database (CARD) captures up-to-date fulllength SARS-CoV-2 sequences of ~50,500 isolates from GISAID, GenBank, CHLA Center for Personalized Medicine, and other sources (as of June 18, 2020). Among which, 49,637 isolates carry at least one variation from the reference genome NC_045512, a total of 6,070 variants and 2,513 haplotypes were detected in at least three isolates independently. Together, they constituted the most likely SARS-CoV-2 variant and haplotype landscapes world-wide currently. Evidence supporting positive (orf3a, orf8, S genes) and purifying (M gene) selections were detected, which warrants further investigation. Most interestingly, we identified 1,583 countryprivate variants from 10,238 isolates (20.6% overall) reported in 48 countries. 807 countryprivate haplotypes, defined as a haplotype shared by at least 5 isolates all of which came from the same country, were identified in in 8,656 isolates from 39 countries. United Kingdom, USA, and Australia had 464, 166 and 32 private haplotypes respectively, comprising 22.4%, 16.6% and 16.4% of the isolates from each country. Together with their descendent and private haplotypes with fewer members, 22,171 (45.8%) isolates carried country-private haplotypes globally. The percentage were 28.2-29.6% in January to March, and rapidly increased to 46.4% and 59.6% in April and May, co-occurring with global travel restrictions. The localization of the variant profiles appeared to be similarly accelerating from 14.2% in March and 28.4% in April to over 40% isolates carrying the country-private variants around May. In summary, a common pattern is seen world-wide in COVID-19 in which at the onset of disease there appeared to be a significant number of SARS-CoV-2 variants that accumulate quickly and then begin to rapidly coalesce into distinct haplotypes. This may be the result of localized outbreaks due to factors such as multiple points viral introduction, geographic separation and the introduction of policies such as travel restriction, social distancing and quarantine, resulting in the emergence of country-private haplotypes.

positive: Multi-Omics and Integrated Network Approach to Unveil Evolutionary Patterns, Mutational Hotspots, Functional Crosstalk and Regulatory Interactions in SARS-CoV-2 SARS-CoV-2 responsible for the pandemic of the Severe Acute Respiratory Syndrome resulting in infections and death of millions worldwide with maximum cases and mortality in USA. The current study focuses on understanding the population specific variations attributing its high rate of infections in specific geographical regions which may help in developing appropriate treatment strategies for COVID-19 pandemic. Rigorous phylogenetic network analysis of 245 complete SARS-CoV-2 genomes inferred five central clades named a (ancestral), b, c, d and e (subtype e1 & e2) showing both divergent and linear evolution types. The clade d & e2 were found exclusively comprising of USA strains with highest known mutations. Clades were distinguished by ten co-mutational combinations in proteins; Nsp3, ORF8, Nsp13, S, Nsp12, Nsp2 and Nsp6 generated by Amino Acid Variations (AAV). Our analysis revealed that only 67.46 % of SNP mutations were carried by amino acid at phenotypic level. T1103P mutation in Nsp3 was predicted to increase the protein stability in 238 strains except six strains which were marked as ancestral type; whereas com (P5731L & Y5768C) in Nsp13 were found in 64 genomes of USA highlighting its 100% co-occurrence. Docking study highlighted mutation (D7611G) caused reduction in binding of Spike proteins with ACE2, but it also showed better interaction with TMPRSS2 receptor which may contribute to its high transmissibility in USA strains. In addition, we found host proteins, MYO5A, MYO5B & MYO5C had maximum interaction with viral hub proteins (Nucleocapsid, Spike & Membrane). Thus, blocking the internalization pathway by inhibiting MYO-5 proteins which could be an effective target for COVID-19 treatment. The functional annotations of the Host-Pathogen Interaction (HPI) network were found to be highly associated with hypoxia and thrombotic conditions confirming the vulnerability and severity of infection in the patients. We also considered the presence of CpG islands in Nsp1 and N proteins which may confers the ability of SARS-CoV-2 to enter and trigger methyltransferase activity inside host cell.

positive: SARS-CoV-2/COVID-19: Viral Genomics, Epidemiology, Vaccines, and Therapeutic Interventions The COVID-19 pandemic is due to infection caused by the novel SARS-CoV-2 virus that impacts the lower respiratory tract. The spectrum of symptoms ranges from asymptomatic infections to mild respiratory symptoms to the lethal form of COVID-19 which is associated with severe pneumonia, acute respiratory distress, and fatality. To address this global crisis, up-to-date information on viral genomics and transcriptomics is crucial for understanding the origins and global dispersion of the virus, providing insights into viral pathogenicity, transmission, and epidemiology, and enabling strategies for therapeutic interventions, drug discovery, and vaccine development. Therefore, this review provides a comprehensive overview of COVID-19 epidemiology, genomic etiology, findings from recent transcriptomic map analysis, viral-human protein interactions, molecular diagnostics, and the current status of vaccine and novel therapeutic intervention development. Moreover, we provide an extensive list of resources that will help the scientific community access numerous types of databases related to SARS-CoV-2 OMICs and approaches to therapeutics related to COVID-19 treatment.

positive: Phylogenetic analysis of SARS-CoV-2 genomes in Turkey COVID-19 has effectively spread worldwide. As of May 2020, Turkey is among the top ten countries with the most cases. A comprehensive genomic characterization of the virus isolates in Turkey is yet to be carried out. Here, we built a phylogenetic tree with globally obtained 15,277 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. We identified the subtypes based on the phylogenetic clustering in comparison with the previously annotated classifications. We performed a phylogenetic analysis of the first 30 SARS-CoV-2 genomes isolated and sequenced in Turkey. We suggest that the first introduction of the virus to the country is earlier than the first reported case of infection. Virus genomes isolated from Turkey are dispersed among most types in the phylogenetic tree. We find 2 of the seventeen subclusters enriched with the isolates of Turkey, which likely have spread expansively in the country. Finally, we traced virus genomes based on their phylogenetic placements. This analysis suggested multiple independent international introductions of the virus and revealed a hub for the inland transmission. We released a web application to track the global and interprovincial virus spread of the isolates from Turkey in comparison to thousands of genomes worldwide.

positive: Molecular Characterization and Amino Acid Homology of Nucleocapsid (N) Protein in SARS-CoV-1, SARS-CoV-2, MERS-CoV, and Bat Coronavirus Coronavirus disease - 2019 (COVID-19) pandemic, due to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is posing a severe bio threat to the entire world Nucleocapsids of SARS-CoV-2 and the related viruses were studied for gene and amino acid sequence homologies In this study, we established similarities and differences in nucleocapsids in SARS-CoV-2, severe acute respiratory syndrome - coronavirus-1 (SARS-CoV-1), bat coronavirus (bat-CoV) and Middle East respiratory syndrome - coronavirus (MERS-CoV) We conducted a detailed analysis of the nucleocapsid protein amino acid and gene sequence encoding it, found in various coronavirus strains After thoroughly screening the different nucleocapsids, we observed a close molecular homology between SARS-CoV-1 and SARS-CoV-2 More than 95% sequence similarity was observed between the two SARS-CoV strains Bat-CoV and SARS-CoV-2 showed 92% sequence similarity MERS-CoV and SARS-CoV-2 nucleocapsid analysis indicated only 65% identity Molecular characterization of nucleocapsids from various coronaviruses revealed that SARS-CoV 2 is more related to SARS-CoV 1 and bat-CoV SARS-CoV 2 exhibited less resemblance with MERS-CoV SARS-CoV 2 showed less similarity to MERS-CoV Thus, either SARS-CoV-1 or bat-CoV may be the source of SARS-CoV-2 evolution Moreover, the existing differences in nucleocapsid molecular structures in SARS-CoV-2 make this virus more virulent and highly infectious, which means that the non-identical SARS-CoV-2 genes (which are absent in SARS-CoV-1 and bat-CoV) are responsible for COVID-19 severity We observed that SARS-CoV-2 nucleocapsid from different locations varied in amino acid sequences This revealed that there are many SARS-CoV-2 subtypes/subsets currently circulating globally This study will help to develop antiviral vaccine and drugs, study viral replication and immunopathogenesis, and synthesize monoclonal antibodies that can be used for precise COVID-19 diagnosis, without false-positive/false-negative results

positive: No evidence for distinct types in the evolution of SARS-CoV-2 A recent study by Tang et al. (2020) claimed that two major types of SARS-CoV-2 had evolved in the ongoing COVID-19 pandemic and that one of these types was more “aggressive” than the other. Given the repercussions of these claims and the intense media coverage of these types of articles, we have examined in detail the data presented by Tang et al, and show that the major conclusions of that paper cannot be substantiated. Using examples from other viral outbreaks we discuss the difficulty in demonstrating the existence or nature of a functional effect of a viral mutation, and we advise against overinterpretation of genomic data during the pandemic.

positive: Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.

positive: Microstructure, pathophysiology and potential therapeutics of COVID-19: A comprehensive review There have been over 7 million cases and almost 413,372 deaths globally due to the novel coronavirus (2019-nCoV) associated disease COVID-19, as of June 11, 2020. Phylogenetic analysis suggests that there is a common source for these infections. The overall sequence similarities between the spike protein of 2019-nCoV and that of SARS-CoV are known to be around 76-78% and 73-76% for whole protein and receptor-binding domain (RBD), respectively. Thus, they have the potential to serve as drug and/ or vaccine candidate. However, the individual response against 2019-nCoV differs due to genetic variations in the human population. Understanding the variations in Angiotensin-converting enzyme 2 (ACE2) and human leukocyte antigen (HLA) that may affect the severity of 2019-nCoV infection could help in identifying individuals at higher risk from the COVID-19. A number of potential drugs/vaccines as well as antibody/cytokine-based therapeutics are running in various developmental stages of preclinical/clinical trials against SARS-CoV, MERS-CoV and 2019-nCoV with substantial cross-reactivity, which may be used against COVID-19. For diagnosis, reverse transcription polymerase chain reaction (RT-PCR) is the gold standard test for initial diagnosis of COVID-19. Kit based on serological tests are also recommended for investigating the spread of COVID-19 but it is challenging due to antibodies cross-reactivity. This review comprehensively summarizes the recent reports available regarding the host-pathogen interaction, morphological and genomic structure of the virus, and the diagnostic techniques as well as available and potential therapeutics against COVID-19. This article is protected by copyright. All rights reserved.

positive: SARS-CoV-2 host diversity: An update of natural infections and experimental evidences Abstract Coronavirus disease-19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is now a pandemic threat. This virus is supposed to be spread by human to human transmission. Cellular angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2 which is identical or similar in different species of animals such as pigs, ferrets, cats, orangutans, monkeys, and humans. Moreover, a recent study predicted that dog might be secondary host during the evolution of SARS-CoV-2 from bat to human. Therefore, there is a possibility of spreading SARS-CoV-2 through domestic pets. There are now many reports of SARS-CoV-2 positive cases in dogs, cats, tigers, lion, and minks. Experimental data showed ferrets and cats are highly susceptible to SARS-CoV-2 as infected by virus inoculation and can transmit the virus directly or indirectly by droplets or airborne route. Based on these natural infection reports and experimental data, whether the pets are responsible for SARS-CoV-2 spread to human; needs to be deeply investigated. Humans showing clinical symptoms of respiratory infections have been undergoing for COVID-19 diagnostic test but many infected people and few pets confirmed with SARS-CoV-2 remained asymptomatic. In this review, we summarize the natural cases of SARS-CoV-2 in animals with the latest researches conducted in this field. This review will be helpful to think insights of SARS-CoV-2 transmissions, spread, and demand for sero-prevalence studies especially in companion animals.

positive: Genomic epidemiology reveals multiple introductions and spread of SARS-CoV-2 in the Indian state of Karnataka Karnataka, a state in south India, reported its first case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on March 8, 2020, more than a month after the first case was reported in India. We used a combination of contact tracing and genomic epidemiology to trace the spread of SARS-CoV-2 in the state up until May 21, 2020 (1578 cases). We obtained 47 full genomes of SARS-CoV-2 which clustered into six lineages (Pangolin lineages-A, B, B.1, B.1.1, B.4, and B.6). The lineages in Karnataka were known to be circulating in China, Southeast Asia, Iran, Europe and other parts of India and are likely to have been imported into the state both by international and domestic travel. Our sequences grouped into 12 contact clusters and 11 cases with no known contacts. We found nine of the 12 contact clusters had a single lineage of the virus, consistent with multiple introductions and most (8/12) were contained within a single district, consistent with local spread. In most of the twelve clusters, the index case (9/12) and spreaders (8/12) were symptomatic. Of the 47 sequences, 31 belonged to the B/B.6 lineage, including seven of eleven cases with no known contact, this is consistent with the ongoing transmission of this lineage in the state. Genomic epidemiology of SARS-CoV-2 in Karnataka is consistent with multiple introductions of the virus followed by local transmission in parallel with ongoing viral evolution. This is the first study from India combining genomic data with epidemiological information emphasizing the need for an integrated approach to outbreak response.

positive: Emergence of Novel Coronavirus and COVID-19: whether to stay or die out? The last century has witnessed several assaults from RNA viruses, resulting in millions of death throughout the world. The 21st century appears no longer an exception, with the trend continued with escalated fear of SARS coronavirus in 2002 and further concern of influenza H5N1 in 2003. A novel influenza virus created the first pandemic of the 21st century, the pandemic flu in 2009 preceded with the emergence of another deadly virus, MERS-CoV in 2012. A novel coronavirus “SARS-CoV-2” (and the disease COVID-19) emerged suddenly, causing a rapid outbreak with a moderate case fatality rate. This virus is continuing to cause health care providers grave concern due to the lack of any existing immunity in the human population, indicating their novelty and lack of previous exposure. The big question is whether this novel virus will be establishing itself in an endemic form or will it eventually die out? Endemic viruses during circulation may acquire mutations to infect naïve, as well as individual with pre-existing immunity. Continuous monitoring is strongly advisable, not only to the newly infected individuals, but also to those recovered individuals who were infected by SARS-CoV-2 as re-infection may lead to the selection of escape mutants and subsequent dissemination to the population.

positive: SARS-CoV-2 molecular and phylogenetic analysis in COVID-19 patients: A preliminary report from Iran. BACKGROUND The aim of the current study was to investigate and track the SARS-CoV-2 in Iranian Coronavirus Disease 2019 (COVID-19) patients using molecular and phylogenetic methods. METHODS We enrolled seven confirmed cases of COVID-19 patients for the phylogenetic assessment of the SARS-CoV-2 in Iran. The nsp-2, nsp-12, and S genes were amplified using one-step RT-PCR and sequenced using Sanger sequencing method. Popular bioinformatics software were used for sequences alignment and analysis as well as phylogenetic construction. RESULTS The mean age of the patients in the present study was 60.42 ± 9.94 years and 57.1% (4/7) were male. The results indicated high similarity between Iranian and Chinese strains. We could not find any particular polymorphisms in the assessed regions of the three genes. Phylogenetic trees by neighbor-joining and maximum likelihood method of nsp-2, nsp-12, and S genes showed that there are not any differences between Iranian isolates and those of other countries. CONCLUSION As a preliminary phylogenetic study in Iranian SARS-CoV-2 isolates, we found that these isolates are closely related to the Chinese and reference sequences. Also, no sensible differences were observed between Iranian isolates and those of other countries. Further investigations are recommended using more comprehensive methods and larger sample sizes.

positive: Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.

positive: Main Routes of Entry and Genomic Diversity of SARS-CoV-2, Uganda. We established rapid local viral sequencing to document the genomic diversity of severe acute respiratory syndrome coronavirus 2 entering Uganda. Virus lineages closely followed the travel origins of infected persons. Our sequence data provide an important baseline for tracking any further transmission of the virus throughout the country and region.

positive: SARS-CoV-2 transmission chains from genetic data: a Danish case study Background The COVID-19 pandemic caused by the SARS-CoV-2 virus started in China in December 2019 and has since spread globally. Information about the spread of the virus in a country can inform the gradual reopening of a country and help to avoid a second wave of infections. Denmark is currently opening up after a lockdown in mid-March. Methods We perform a phylogenetic analysis of 742 publicly available Danish SARS-CoV-2 genome sequences and put them into context using sequences from other countries. Result Our findings are consistent with several introductions of the virus to Denmark from independent sources. We identify several chains of mutations that occurred in Denmark and in at least one case find evidence that the virus spread from Denmark to other countries. A number of the mutations found in Denmark are non-synonymous, and in general there is a considerable variety of strains. The proportions of the most common haplotypes is stable after lockdown. Conclusion Our work shows how genetic data can be used to identify routes of introduction of a virus into a region and provide alternative means for verifying existing assumptions. For example, our analysis supports the hypothesis that the virus was brought to Denmark by skiers returning from Ischgl. On the other hand, we identify transmission chains suggesting that Denmark was part of a network of countries among which the virus was being transmitted; thus challenging the common narrative that Denmark only got infected from abroad. Our analysis does not indicate that the major haplotypes appearing in Denmark have a different degree of virality. Our methods can be applied to other countries, regions or even highly localised outbreaks. When used in real-time, we believe they can serve to identify transmission events and supplement traditional methods such as contact tracing.

positive: Evolutionary relationships and sequence-structure determinants in human SARS coronavirus-2 spike proteins for host receptor recognition Coronavirus disease 2019 (COVID-19) is a pandemic infectious disease caused by novel severe acute respiratory syndrome coronavirus-2 (SARS CoV-2). The SARS CoV-2 is transmitted more rapidly and readily than SARS CoV. Both, SARS CoV and SARS CoV-2 via their glycosylated spike proteins recognize the human angiotensin converting enzyme-2 (ACE-2) receptor. We generated multiple sequence alignments and phylogenetic trees for representative spike proteins of SARS CoV and SARS CoV-2 from various host sources in order to analyze the specificity in SARS CoV-2 spike proteins required for causing infection in humans. Our results show that among the genomes analyzed, two sequence regions in the N-terminal domain "MESEFR" and "SYLTPG" are specific to human SARS CoV-2. In the receptor-binding domain, two sequence regions "VGGNY" and "EIYQAGSTPCNGV" and a disulfide bridge connecting 480C and 488C in the extended loop are structural determinants for the recognition of human ACE-2 receptor. The complete genome analysis of representative SARS CoVs from bat, civet, human host sources, and human SARS CoV-2 identified the bat genome (GenBank code: MN996532.1) as closest to the recent novel human SARS CoV-2 genomes. The bat SARS CoV genomes (GenBank codes: MG772933 and MG772934) are evolutionary intermediates in the mutagenesis progression toward becoming human SARS CoV-2.

positive: A genome epidemiological study of SARS-CoV-2 introduction into Japan Background: After the first case of COVID-19 in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases appeared until early April, many with unclear infection routes exhibiting no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases until early April and characterise the genealogical networks to demonstrate possible routes of spread in Japan. Methods: Nasopharyngeal specimens were collected from patients and a quantitative reverse transcription polymerase chain reaction testing for SARS-CoV-2 was performed. Positive RNA samples were subjected whole genome sequencing and a haplotype network analysis was performed. Findings: Some of the primary clusters identified during January and February in Japan directly descended from Wuhan-Hu-1-related isolates in China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that COVID-19 cases from late March through early April may have caused an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. National self-restraint during February was effective in mitigating the COVID-19 spread, but late action on stopping immigration and declaring national emergency in Japan might be involved in the later increase in cases. Interpretation: Genome surveillance suggested that at least two distinct SARS-CoV-2 introductions from China and other countries occurred. Funding: Japan Agency for Medical Research and Development.

positive: Comparison of the COVID-2019 (SARS-CoV-2) pathogenesis with SARS-CoV and MERS-CoV infections The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in several patients who traveled to Wuhan or went to a seafood wholesale market in Wuhan. The phylogenetic tree showed that SARS-CoV-2 was 96.2% identical to bat β-coronaviruses from lineage B. Also, several studies reported that SARS-CoV-2 uses the SARS-CoV receptor, angiotensin-converting enzyme 2, for entry to target cells. Lung alveolar and small intestine are potential targets for SARS-CoV-2 due to the high expression of the angiotensin-converting enzyme 2 receptor. In this review, we focused on the zoonotic β-coronaviruses and given there is no specific drug or vaccine for coronavirus disease 2019, we reviewed the literature on the therapy options for SARS and Middle East respiratory syndrome coronavirus infection, in order to discover their possible use in the treatment of SARS-CoV-2 infections.

positive: Betacoronavirus Genomes: How Genomic Information has been Used to Deal with Past Outbreaks and the COVID-19 Pandemic. In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of coronaviruses in the aftermath of the 2002-2003 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-2020 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2. We focus on shared genomic features of the betacoronaviruses and the application of genomic information to phylogenetic analysis, molecular epidemiology and the design of diagnostic systems, potential drugs and vaccine candidates.

positive: Climatic-niche evolution of SARS CoV-2 Adaptation of species to new environments is governed by natural selection that discriminates among genetic variations and favors survival of the fittest. Here, we propose climate plays an important role in the evolution of SARS CoV-2 and the spread of COVID-19 all over the world which was previously not known. To understand the climatic factors responsible for shaping the molecular determinants of the novel coronavirus, genotyping SARS CoV-2 across different latitudes and Koppen’s climate is imperative. It seems this virus follows inverse latitudinal biodiversity gradient due to its preference towards Koppen’s temperate (C) and cold climate (D). Our molecular phylogenetic analysis revealed division of 176 SARS CoV-2 strains into two variant groups, G1 and G2, well defined by four mutations. Initially, SARS CoV-2 was restricted to a “humid-subtropical” (Cfa) climate of southeast China, which soon spread all over the world having C climate. Genomic information superimposed on global Koppen’s climate map elucidates that the gradation “humid-subtropical” (Cfa) and “marine-temperate” (Cfb) to “humid-continental” (Dfa-Dfb) climate drives the evolution of G1 into G2 variant group. It seems an early infection in Europe and USA is due to the dominance of C climate. Russia and North America were infected through linkage of C to D climate and South America from C to A climate. Our study elucidates viruses are sensitive to climate and combined genomic and climatic studies provide crucial information about the pathogenesis and natural spreading pathways during a pandemic which will enable us to take pre-emptive precautionary measures in such outbreaks. Graphical Abstract In Brief The authors elucidate adaptation of SARS CoV-2 to different climates by studying phylogenetics and the distribution of strains on Koppen’s climate map. Highlights SARS CoV-2 follows inverse latitudinal gradient. Phylogenetic network divides SARS CoV-2 strains into two variant groups, G1 and G2. G1 strains is restricted to Koppen’s “temperate” climate (mainly Cfa-Cfb). G2 strains has evolved from G1 to sustain in “humid-continental” (Dfa-Dfb) and “tropical-savannah” (Aw) climate.

positive: Comprehensive evolution and molecular characteristics of a large number of SARS-CoV-2 genomes revealed its epidemic trend and possible origins Objectives To reveal epidemic trend and possible origins of SARS-CoV-2 by exploring its evolution and molecular characteristics based on a large number of genomes since it has infected millions of people and spread quickly all over the world. Methods Various evolution analysis methods were employed. Results The estimated Ka/Ks ratio of SARS-CoV-2 is 1.008 or 1.094 based on 622 or 3624 SARS-CoV-2 genomes, and the time to the most recent common ancestor (tMRCA) was inferred in late September 2019. Further 9 key specific sites of highly linkage and four major haplotypes H1, H2, H3 and H4 were found. The Ka/Ks, detected population size and development trends of each major haplotype showed H3 and H4 subgroups were going through a purify evolution and almost disappeared after detection, indicating H3 and H4 might have existed for a long time, while H1 and H2 subgroups were going through a near neutral or neutral evolution and globally increased with time. Notably the frequency of H1 was generally high in Europe and correlated to death rate (r>0.37). Conclusions In this study, the evolution and molecular characteristics of more than 16000 genomic sequences provided a new perspective for revealing epidemiology of SARS-CoV-2.

positive: A comparative analysis for SARS-CoV-2 COVID-19 has affected the world tremendously. It is critical that biological experiments and clinical designs are informed by computational approaches for time- and cost-effective solutions. Comparative analyses particularly can play a key role to reveal structural changes in proteins due to mutations, which can lead to behavioural changes, such as the increased binding of the SARS-CoV-2 surface glycoprotein to human ACE2 receptors. The aim of this report is to provide an easy to follow tutorial for biologists and others without delving into different bioinformatics tools. More complex analyses such as the use of large-scale computational methods can then be utilised. Starting with a SARS-CoV-2 genome sequence, the report shows visualising DNA sequence features, deriving amino acid sequences, and aligning different genomes to analyse mutations and differences. The report provides further insights into how the SARS-CoV-2 surface glycoprotein mutated for higher binding affinity to human ACE2 receptors, compared to the SARS-CoV protein, by integrating existing 3D protein models.

positive: CovProfile: profiling the viral genome and gene expressions of SARS-COV-2 The SARS-CoV-2 virus has infected more than one million people worldwide to date. Knowing its genome and gene expressions is essential to understand the virus’ mechanism. Here, we propose a computational tool CovProfile to detect the viral genomic variations as well as viral gene expressions from the sequences obtained from Nanopore devices. We applied CovProfile to 11 samples, each from a terminally ill patient, and discovered that all the patients are infected by multiple viral strains, which might affect the reliability of phylogenetic analysis. Moreover, the expression of viral genes ORF1ab gene, S gene, M gene, and N gene are high among most of the samples. While performing the tests, we noticed a consistent abundance of transcript segments of MUC5B, presumably from the host, across all the samples.

positive: Complete Genome Sequence of a Novel Coronavirus (SARS-CoV-2) Isolate from Bangladesh The complete genome sequence of a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) isolate obtained from a nasopharyngeal swab from a patient with COVID-19 in Bangladesh is reported.

positive: The 2019-new coronavirus epidemic: Evidence for virus evolution There is a worldwide concern about the new coronavirus 2019-nCoV as a global public health threat. In this article, we provide a preliminary evolutionary and molecular epidemiological analysis of this new virus. A phylogenetic tree has been built using the 15 available whole genome sequences of 2019-nCoV, 12 whole genome sequences of 2019-nCoV, and 12 highly similar whole genome sequences available in gene bank (five from the severe acute respiratory syndrome, two from Middle East respiratory syndrome, and five from bat SARS-like coronavirus). Fast unconstrained Bayesian approximation analysis shows that the nucleocapsid and the spike glycoprotein have some sites under positive pressure, whereas homology modeling revealed some molecular and structural differences between the viruses. The phylogenetic tree showed that 2019-nCoV significantly clustered with bat SARS-like coronavirus sequence isolated in 2015, whereas structural analysis revealed mutation in Spike Glycoprotein and nucleocapsid protein. From these results, the new 2019-nCoV is distinct from SARS virus, probably trasmitted from bats after mutation conferring ability to infect humans.

positive: In silico studies on the comparative characterization of the interactions of SARS‐CoV‐2 spike glycoprotein with ACE‐2 receptor homologs and human TLRs Coronavirus disease‐2019 (COVID‐19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection has come out as a major threat for mankind in recent times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS‐CoV‐2), its mode of interaction with the angiotensin‐converting enzyme‐2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll‐like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS‐CoV‐2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS‐CoV‐2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS‐CoV‐2 from a bat and also revealed that TLR4 may have a crucial role in the virus‐induced inflammatory consequences associated with COVID‐19. Therefore, selective targeting of TLR4‐spike protein interaction by designing competitive TLR4‐antagonists could pave a new way to treat COVID‐19. Finally, this study is expected to improve our understanding on the immunobiology of SARS‐CoV‐2 and could be useful in adopting spike protein, ACE2, or TLR‐guided intervention strategy against COVID‐19 shortly.

positive: Defining the Pandemic at the State Level: Sequence-Based Epidemiology of the SARS-CoV-2 virus by the Arizona COVID-19 Genomics Union (ACGU) In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China causing severe morbidity and mortality. Since then, the virus has swept across the globe causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, provided to us by the Arizona Department of Health Services, and at Arizona State University and the University of Arizona, collected as part of community surveillance projects. Phylogenetic analysis of 79 genomes we generated from across Arizona revealed a minimum of 9 distinct introductions throughout February and March. We show that >80% of our sequences descend from clades that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related cases in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020.

positive: First detection and genome sequencing of SARS‐CoV‐2 in an infected cat in France After its first description in Wuhan (China), SARS‐CoV‐2 the agent of coronavirus disease 2019 (COVID‐19) rapidly spread worldwide. Previous studies suggested that pets could be susceptible to SARS‐CoV‐2. Here, we investigated the putative infection by SARS‐CoV‐2 in 22 cats and 11 dogs from owners previously infected or suspected of being infected by SARS‐CoV‐2. For each animal, rectal, nasopharyngeal swabs and serum were taken. Swabs were submitted to RT‐qPCR assays targeting 2 genes of SARS‐CoV‐2. All dogs were tested SARS‐CoV‐2 negative. One cat was tested positive by RT‐qPCR on rectal swab. Nasopharyngeal swabs from this animal were tested negative. This cat showed mild respiratory and digestive signs. Serological analysis confirms the presence of antibodies against the SARS‐CoV‐2 in the both serum samples taken 10 days apart. Genome sequence analysis revealed that the cat SARS‐CoV‐2 belongs to the phylogenetic clade A2a like most of the French human SARS‐CoV‐2. This study reports for the first time the natural infection of a cat in France (near Paris) probably through their owners. There is currently no evidence that cats can spread COVID‐19 and owners should not abandon their pets or compromise their welfare.

positive: Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

positive: RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881â nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into ß-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370â nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.

positive: A candidate multi-epitope vaccine against SARS-CoV-2 In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.

positive: Insurgence and worldwide diffusion of genomic variants in SARS-CoV-2 genomes The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.

positive: Insights into The Codon Usage Bias of 13 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Isolates from Different Geo-locations Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus disease 2019 (COVID-19) which is an infectious disease that spread throughout the world and was declared as a pandemic by the World Health Organization (WHO). In the present study, we analyzed genome-wide codon usage patterns in 13 SARS-CoV-2 isolates from different geo-locations (countries) by utilizing different CUB measurements. Nucleotide and di-nucleotide compositions displayed bias toward A/U content in all codon positions and CpU-ended codons preference, respectively. Relative Synonymous Codon Usage (RSCU) analysis revealed 8 common putative preferred codons among all the 13 isolates. Interestingly, all of the latter codons are A/U-ended (U-ended: 7, A-ended: 1). Cluster analysis (based on RSCU values) was performed and showed comparable results to the phylogenetic analysis (based on their whole genome sequences) indicating that the CUB pattern may reflect the evolutionary relationship between the tested isolates. To investigate the force (mutation and/or selection) influencing the pattern of CUB in SARS-CoV-2 coding sequences, we employed the following; (i). Effective number of codons (ENc), (ii). ENc-GC3 plot, (iii). Neutrality plot, and (iv) Codon adaptation index (CAI). According to their results, natural selection and/or other factors (not investigated in this study) may be the dominant force driving SARS-CoV-2 CUB. It is also worth mentioning that, by using the most expressed genes in human lung tissues as a reference set, some viral genes such as Nucleocapsid phosphoprotein, ORF7a protein, and surface glycoprotein had high CAI values which may indicate for selection force acting on their codon usage, as they play important roles in viral assembly and may help viruses avoid the host immune system. The outcome of our study may help in understanding the underlying factors involved in the evolution of SARS-CoV-2 viruses, and the interactions with their host. Also, it may aid in vaccine design strategies.

positive: Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China An in-depth annotation of the newly discovered coronavirus (2019-nCoV) genome has revealed differences between 2019-nCoV and severe acute respiratory syndrome (SARS) or SARS-like coronaviruses. A systematic comparison identified 380 amino acid substitutions between these coronaviruses, which may have caused functional and pathogenic divergence of 2019-nCoV.

positive: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): a review In recent years, the prevalence and spread of coronavirus has had a huge impact on global public health. Due to the incomplete understanding of the pathogenic mechanism of the virus, it is difficult for humans to fight against the virus quickly and effectively once the outbreak occurs. In early 2020, a novel coronavirus was discovered in Wuhan, China. Soon after, similar cases were found in other countries around the world, and the number of infected people increased rapidly. So far, the global cumulative number of infected people has exceeded 3 million, and more than 200,000 people have died, which has had a huge impact on global human health and economic development. Every outbreak of disease makes a deep impression on mankind. Herein, we summarize the virology, epidemiology, clinical manifestations, diagnosis, treatment and prevention of SARS-CoV-2, and hope that countries can control the outbreak as soon as possible to minimize the loss.

positive: Phylogenetic perspectives on the epidemiology and origins of SARS and SARS-like coronaviruses Abstract Severe Acute Respiratory Syndrome (SARS) is a respiratory disease caused by a zoonotic coronavirus (CoV) named SARS-CoV (SCoV), which rapidly swept the globe after its emergence in rural China during late 2002. The origins of SCoV have been mysterious and controversial, until the recent discovery of SARS-like CoV (SLCoV) in bats and the proposal of bats as the natural reservior of the Coronaviridae family. In this article, we focused on discussing how phylogenetics contributed to our understanding towards the emergence and transmission of SCoV. We first reviewed the epidemiology of SCoV from a phylogenetic perspective and discussed the controversies over its phylogenetic origins. Then, we summarized the phylogenetic findings in relation to its zoonotic origins and the proposed inter-species viral transmission events. Finally, we also discussed how the discoveries of SCoV and SLCoV expanded our knowledge on the evolution of the Coronaviridae family as well as its implications on the possible future re-emergence of SCoV.

positive: Phylogenetic Analysis and Structural Perspectives of RNA-Dependent RNA-Polymerase Inhibition from SARs-CoV-2 with Natural Products ABSTRACT: Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements. GRAPHIC ABSTRACT: [Image: see text]

positive: SARS-CoV-2 genomic surveillance in Taiwan revealed novel ORF8-deletion mutant and clade possibly associated with infections in Middle East. Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

positive: Functional pangenome analysis suggests inhibition of the protein E as a readily available therapy for COVID-2019 The spread of the novel coronavirus (SARS-CoV-2) has triggered a global emergency, that demands urgent solutions for detection and therapy to prevent escalating health, social and economic impacts. The spike protein (S) of this virus enables binding to the human receptor ACE2, and hence presents a prime target for vaccines preventing viral entry into host cells1. The S proteins from SARS-CoV-1 and SARS-CoV-2 are similar2, but structural differences in the receptor binding domain (RBD) preclude the use of SARS-CoV-1–specific neutralizing antibodies to inhibit SARS-CoV-23. Here we used comparative pangenomic analysis of all sequenced Betacoronaviruses to reveal that, among all core gene clusters present in these viruses, the envelope protein E shows a variant shared by SARS and SARS-Cov2 with two completely-conserved key functional features, an ion-channel and a PDZ-binding Motif (PBM). These features trigger a cytokine storm that activates the inflammasome, leading to increased edema in lungs causing the acute respiratory distress syndrome (ARDS)4-6, the leading cause of death in SARS-CoV-1 and SARS-CoV-2 infection7,8. However, three drugs approved for human use may inhibit SARS-CoV-1 and SARS-CoV-2 Protein E, either acting upon the ion channel (Amantadine and Hexamethylene amiloride9,10) or the PBM (SB2035805), thereby potentially increasing the survival of the host, as already demonstrated for SARS-CoV-1in animal models. Hence, blocking the SARS protein E inhibits development of ARDS in vivo. Given that our results demonstrate that the protein E subcluster for the SARS clade is quasi-identical for the key functional regions of SARS-CoV-1 and SARS-CoV-2, we conclude that use of approved drugs shown to act as SARS E protein inhibitors can help prevent further casualties from COVID-2019 while vaccines and other preventive measures are being developed.

positive: Genomic characterization and phylogenetic analysis of SARS‐COV‐2 in Italy This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) isolated from three patients involved in the first outbreak of COVID‐19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS‐CoV‐2 was present in Italy weeks before the first reported cases of infection.

positive: COVID-19 in Latin America: Contrasting phylodynamic inference with epidemiological surveillance. Introduction: SARS-CoV-2 infection has represented the one of the largest challenges for humanity. This virus was first detected in Latin America and the Caribbean (LAC) in Brazil in February 26, 2020 and it has revealed important gaps in infectious disease surveillance that must be covered. Phylodynamic analysis is a tool that can help to monitor and adapt traditional surveillance measures in order to cover those. Therefore, this work aims to contrast data driven from epidemiologic surveillance in LAC with parameters inferred from phylodynamic analysis of reported genomes of SARS-CoV-2 across different LAC countries Methods: We obtained epidemiological data from daily reports provided by European Centre for Disease Prevention and Control up to 13th May, 2020. We estimated Effective Reproductive Number (Re) and calculated epidemic curves with exponential growth (EG) and maximum likelihood (ML) methods. SARS-CoV-2 phylodynamic in Latin-American was analyzed using sequences reported in GISAID for Central and South America up to May 13th 2020. Sequences were aligned, and ML phylogeny was constructed. Coalescent model Birth Death SIR (serial) was run, and SIR trajectories from the birth-death SIR model were plotted. Results: A total of 404,448 cases were reported up to 13th May 2020. Overall reproduction number for Latin America, estimated through the EG and ML methods, were 1.424 (IC95% 1.422 to 1.426) and 1.305 (IC95% 1.299 to 1.311) respectively. Phylodynamic analysis for Latin America showed an overall Re of 1.27 (IC95% 1.07 to 1.49). We did not find statistically significant differences between epidemiological and phylodynamic data at the cut-off time, except for Brazil. Discussion: Our results support that epidemiological and genomic surveillance are two complementary approaches. Evidence suggests that even with a low number of sequences proper estimations of Re could be performed. We suggest that countries, especially developing countries, should consider to add genomic surveillance to their systems for monitoring and adapting epidemiological surveillance of SARS-CoV-2.

positive: A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV The outbreak of COVID-19 caused by SARS-CoV-2 virus has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore determined the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein to 3.1 Å. CR3022 targets a highly conserved epitope, distal from the receptor-binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBD on the trimeric S protein are in the “up” conformation and slightly rotated. Overall, this study provides molecular insights into antibody recognition of SARS-CoV-2.

positive: Coronavirus phylogeny based on 2D graphical representation of DNA sequence A novel coronavirus has been identified as the cause of the outbreak of severe acute respiratory syndrome (SARS). Previous phylogenetic analyses based on sequence alignments show that SARS‐CoVs form a new group distantly related to the other three groups of previously characterized coronaviruses. In this aritcle, a new approach based on the 2D graphical representation of the whole genome sequence is proposed to analyze the phylogenetic relationships of coronaviruses. The evolutionary distances are obtained through measuring the differences among the two‐dimensional curves. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1196–1202, 2006

positive: The COVID-19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control A pneumonia outbreak with unknown etiology was reported in Wuhan, Hubei province, China, in December 2019, associated with the Huanan Seafood Wholesale Market. The causative agent of the outbreak was identified by the WHO as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), producing the disease named coronavirus disease-2019 (COVID-19). The virus is closely related (96.3%) to bat coronavirus RaTG13, based on phylogenetic analysis. Human-to-human transmission has been confirmed even from asymptomatic carriers. The virus has spread to at least 200 countries, and more than 1,700,000 confirmed cases and 111,600 deaths have been recorded, with massive global increases in the number of cases daily. Therefore, the WHO has declared COVID-19 a pandemic. The disease is characterized by fever, dry cough, and chest pain with pneumonia in severe cases. In the beginning, the world public health authorities tried to eradicate the disease in China through quarantine but are now transitioning to prevention strategies worldwide to delay its spread. To date, there are no available vaccines or specific therapeutic drugs to treat the virus. There are many knowledge gaps about the newly emerged SARS-CoV-2, leading to misinformation. Therefore, in this review, we provide recent information about the COVID-19 pandemic. This review also provides insights for the control of pathogenic infections in humans such as SARS-CoV-2 infection and future spillovers.

positive: Genomic surveillance and phylogenetic analysis reveal multiple introductions of SARS-CoV-2 into a global travel hub in the Middle East International travel played a significant role in the early global spread of SARS-CoV-2. Understanding transmission patterns from different regions of the world will further inform global dynamics of the pandemic. Using data from Dubai in the United Arab Emirates (UAE), a major international travel hub in the Middle East, we establish SARS-CoV-2 full genome sequences from the index and early COVID-19 patients in the UAE. The genome sequences are analyzed in the context of virus introductions, chain of transmissions, and possible links to earlier strains from other regions of the world. Phylogenetic analysis showed multiple spatiotemporal introductions of SARS-CoV-2 into the UAE from Asia, Europe, and the Middle East during the early phase of the pandemic. We also provide evidence for early community-based transmission and catalogue new mutations in SARS-CoV-2 strains in the UAE. Our findings contribute to the understanding of the global transmission network of SARS-CoV-2.

positive: Identification of the hyper-variable genomic hotspot for the novel coronavirus SARS-CoV-2

positive: SARS-CoV-2 and ORF3a: Nonsynonymous Mutations, Functional Domains, and Viral Pathogenesis The effect of the rapid accumulation of nonsynonymous mutations on the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet known. The 3a protein is unique to SARS-CoV and is essential for disease pathogenesis. Our study aimed at determining the nonsynonymous mutations in the 3a protein in SARS-CoV-2 and determining and characterizing the protein’s structure and spatial orientation in comparison to those of 3a in SARS-CoV. A total of 51 different nonsynonymous amino acid substitutions were detected in the 3a proteins among 2,782 SARS-CoV-2 strains. We observed microclonality within the ORF3a gene tree defined by nonsynonymous mutations separating the isolates into distinct subpopulations. We detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. The functional domains were linked to virulence, infectivity, ion channel formation, and virus release. Our study showed the importance of conserved functional domains across the species barrier and revealed the possible role of the 3a protein in the viral life cycle. Observations reported in this study merit experimental confirmation. IMPORTANCE At the surge of the coronavirus disease 2019 (COVID-19) pandemic, we detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. Our analysis showed that the functional domains were linked to virulence, infectivity, ion channel formation, and virus release in SARS-CoV-2 3a. Our study also revealed the functional importance of conserved domains across the species barrier. Observations reported in this study merit experimental confirmation.

positive: Cross‐species transmission of the newly identified coronavirus 2019‐nCoV The current outbreak of viral pneumonia in the city of Wuhan, China, was caused by a novel coronavirus designated 2019‐nCoV by the World Health Organization, as determined by sequencing the viral RNA genome. Many initial patients were exposed to wildlife animals at the Huanan seafood wholesale market, where poultry, snake, bats, and other farm animals were also sold. To investigate possible virus reservoir, we have carried out comprehensive sequence analysis and comparison in conjunction with relative synonymous codon usage (RSCU) bias among different animal species based on the 2019‐nCoV sequence. Results obtained from our analyses suggest that the 2019‐nCoV may appear to be a recombinant virus between the bat coronavirus and an origin‐unknown coronavirus. The recombination may occurred within the viral spike glycoprotein, which recognizes a cell surface receptor. Additionally, our findings suggest that 2019‐nCoV has most similar genetic information with bat coronovirus and most similar codon usage bias with snake. Taken together, our results suggest that homologous recombination may occur and contribute to the 2019‐nCoV cross‐species transmission.

positive: Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CLpro targeting repurposed drug candidates BACKGROUND: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. METHODS: For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CLpro) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. RESULTS: Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CLpro enzyme, which regulates viral replication machinery. CONCLUSIONS: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

positive: Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event Abstract Background A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. Methods Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. Results Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat_SARS-like coronavirus sequences. Specifically, in the 5′-part spanning the first 11,498 nucleotides and the last 3′-part spanning 24,341–30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat_SARS-like coronavirus sequences, whereas in the middle region spanning the 3′-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. Conclusions The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

positive: Introductions and early spread of SARS-CoV-2 in the New York City area New York City (NYC) has emerged as one of the epicenters of the current SARS-CoV2 pandemic. To identify the early events underlying the rapid spread of the virus in the NYC metropolitan area, we sequenced the virus causing COVID19 in patients seeking care at the Mount Sinai Health System. Phylogenetic analysis of 84 distinct SARS-CoV2 genomes indicates multiple, independent but isolated introductions mainly from Europe and other parts of the United States. Moreover, we find evidence for community transmission of SARS-CoV2 as suggested by clusters of related viruses found in patients living in different neighborhoods of the city.

positive: Presence and vitality of SARS-CoV-2 virus in wastewaters and rivers Wastewater-based epidemiology has been proposed to monitor the diffusion and trend of SARS-CoV-2 pandemic. In the present study, raw and treated samples from three wastewater treatment plants, and two river samples characterized the Milano Metropolitan Area, Italy, were surveyed for SARS-CoV-2 RNA positivity to real time PCR and infectiveness. Moreover, whole genome sequencing and phylogenetic analysis of isolated strains was performed. Raw wastewater samples resulted positive to PCR amplification, while treated water samples were always negative (four and two samples, respectively, sampled in two dates). Moreover, the rate of positivity in raw wastewater samples decreased after eight days, in congruence with the epidemiological trend estimated for the interested provinces. Virus infectiveness was always not significant, indicating the effectiveness of wastewater treatments, or the natural decay of viral vitality, which implied the absence of significant risk of infection from wastewaters. Samples from receiving rivers (two sites, sampled in the same dates as wastewaters) showed in some cases a positivity to PCR amplification, probably due to non-treated discharges, or the combined sewage overflows. Nevertheless, also for rivers vitality was negligible, indicating the absence of sanitary risks. Phylogenetic analysis of genome indicated that the isolated virus belongs to the most spread strain present in Europe and similar to another strain found in Lombardy.

positive: A new coronavirus associated with human respiratory disease in China Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health(1–3). Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing(4) of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China(5). This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

positive: Molecular conservation and differential mutation on ORF3a gene in Indian SARS-CoV2 genomes A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory protein in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty-two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2's virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservation of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.

positive: Severe Acute Respiratory Syndrome Historical, Epidemiologic, and Clinical Features Severe acute respiratory syndrome coronavirus (SARS-CoV), emerged from China and rapidly spread worldwide. Over 8098 people fell ill and 774 died before the epidemic ended in July 2003. Bats are likely an important reservoir for SARS-CoV. SARS-like CoVs have been detected in horseshoe bats and civet cats. The main mode of transmission of SARS-CoV is through inhalation of respiratory droplets. Faeco-oral transmission has been recorded. Strict infection control procedures with respiratory and contact precautions are essential. Fever and respiratory symptoms predominate, and diarrhea is common. Treatment involves supportive care. There are no specific antiviral treatments or vaccines available.

positive: Response to Carletti et al, “About the origin of the first two SARS‐CoV‐2 infections in Italy: Inference not supported by appropriate sequence analysis” The first one, as the authors should know, that in order to estimate phylogenies on a natural timescale a 'molecular clock' analysis has to be applied. A molecular clock is a theory attributed to Emile Zuckerkandl and Linus Pauling (1962), describing the relationship between observed genetic distances and time. This article is protected by copyright. All rights reserved.

positive: Decoding the lethal effect of SARS-CoV-2 (novel coronavirus) strains from global perspective: molecular pathogenesis and evolutionary divergence Background COVID-19 is a disease with global public health emergency that have shook the world since its’ first detection in China in December, 2019. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is the pathogen responsible behind this pandemic. The lethality of different viral strains is found to vary in different geographical locations but the molecular mechanism is yet to be known. Methods Available data of whole genome sequencing of different viral strains published by different countries were retrieved and then analysed using Multiple Sequence Alignment and Pair-wise Sequence Alignment leading to Phylogenetic tree construction. Each location and the corresponding genetic variations were screened in depth. Then the variations are analysed at protein level giving special emphasis on Non Synonymous amino acid substitutions. The fatality rates in different countries were matched against the mutation number, rarity of the nucleotide alterations and functional impact of the Non Synonymous changes at protein level, separately and in combination. Findings All the viral strains have been found to evolve from the viral strain of Taiwan (MT192759) which is 100% identical with the ancestor SARS-CoV-2 sequences of Wuhan (NC 045512.2; submitted on 5th Jan, 2020). Transition from C to T (C>T) is the most frequent mutation in this viral genome and mutations A>T, G>A, T>A are the rarest ones, found in countries with maximum fatality rate i.e Italy, Spain and Sweden. 20 Non Synonymous mutations are located in viral genome spanning Orf1ab polyprotein, Surface glycoprotein, Nucleocapsid protein etc. The functional effect on the structure and function of the protein can favourably or unfavourably interact with the host body. Interpretation The fatality outcome depends on three important factors (a) number of mutation (b) rarity of the allelic variation and (c) functional consequence of the mutation at protein level. The molecular divergence, evolved from the ancestral strain (S) lead to extremely lethal (E), lethal(L) and non lethal (N) strains with the involvement of an Intermediate strain(I).

positive: Interplay between co-divergence and cross-species transmission in the evolutionary history of bat coronaviruses Abstract Coronaviruses (CoVs) have been documented in almost every species of bat sampled. Bat CoVs exhibit both extensive genetic diversity and a broad geographic range, indicative of a long-standing host association. Despite this, the respective roles of long-term virus-host co-divergence and cross-species transmission (host-jumping) in the evolution of bat coronaviruses are unclear. Using a phylogenetic approach we provide evidence that CoV diversity in bats is shaped by both species richness and their geographical distribution, and that CoVs exhibit clustering at the level of bat genera, with these genus-specific clusters largely associated with distinct CoV species. Co-phylogenetic analyses revealed that cross-species transmission has been more common than co-divergence across coronavirus evolution as a whole, and that cross-species transmission events were more likely between sympatric bat hosts. Notably, however, an analysis of the CoV RNA polymerase phylogeny suggested that many such host-jumps likely resulted in short-term spill-over infections, with little evidence for sustained onward transmission in new co-roosting host species.

positive: An update on origin of SARS-CoV-2: Despite closest identity, bat (RaTG13) and Pangolin derived Coronaviruses varied in the critical binding site and O-linked glycan residues The initial cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) occurred in Wuhan, China, in December 2019 and swept the world by 23 June 2020 with 8,993,659 active cases, 469,587 deaths across 216 countries, areas or territories. This strongly implies global transmission occurred before the lockdown of China. However, the initial source's transmission routes of SARS-CoV-2 remain obscure and controversial. Research data suggest bat (RaTG13) and pangolin carried CoV were the proximal source of SARS-CoV-2. In this study, we used systematic phylogenetic analysis of Coronavirinae subfamily along with wild type human SARS-CoV, MERS-CoV, and SARS-CoV-2 strains. The key residues of the receptor-binding domain (RBD) and O-linked glycan were compared. SARS-CoV-2 strains were clustered with RaTG13 (97.41% identity), Pangolin-CoV (92.22% identity) and Bat-SL-CoV (80.36% identity), forms a new clade-2 in lineage B of beta-CoV. The alignments of RBD contact residues to ACE2 justified? Those SARS-CoV-2 strains sequences were 100% identical by each other, significantly varied in RaTG13 and pangolin-CoV. SARS-CoV-2 has a polybasic cleavage site with an inserted sequence of PRRA compared to RaTG13 and only PRR to pangolin. Only serine (Ser) in pangolin and both threonine (Thr) and serine (Ser) O-linked glycans were seen in RaTG13, suggesting that a detailed study needed in Pangolin (Manis javanica) and bat (Rhinolophus affinis) related CoV. This article is protected by copyright. All rights reserved.

positive: Rampant C->U hypermutation in the genomes of SARS-CoV-2 and other coronaviruses – causes and consequences for their short and long evolutionary trajectories The pandemic of SARS coronavirus 2 (SARS-CoV-2) has motivated an intensive analysis of its molecular epidemiology following its worldwide spread. To understand the early evolutionary events following its emergence, a dataset of 985 complete SARS-CoV-2 sequences was assembled. Variants showed a mean 5.5-9.5 nucleotide differences from each other, commensurate with a mid-range coronavirus substitution rate of 3×10−4 substitutions/site/year. Almost half of sequence changes were C->U transitions with an 8-fold base frequency normalised directional asymmetry between C->U and U->C substitutions. Elevated ratios were observed in other recently emerged coronaviruses (SARS-CoV and MERS-CoV) and to a decreasing degree in other human coronaviruses (HCoV-NL63, -OC43, -229E and -HKU1) proportionate to their increasing divergence. C->U transitions underpinned almost half of the amino acid differences between SARS-CoV-2 variants, and occurred preferentially in both 5’U/A and 3’U/A flanking sequence contexts comparable to favoured motifs of human APOBEC3 proteins. Marked base asymmetries observed in non-pandemic human coronaviruses (U>>A>G>>C) and low G+C contents may represent long term effects of prolonged C->U hypermutation in their hosts. Importance The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short and long term evolution. Repeated cycles of mutation and reversion in favoured mutational hotspots and the widespread occurrence of amino acid changes with no adaptive value for the virus represents a quite different paradigm of virus sequence change from neutral and Darwinian evolutionary frameworks that are typically used in molecular epidemiology investigations.

positive: Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India. Background & objectives : Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019. Methods : Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp. Results : Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1. Interpretation & conclusions : This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

positive: A previously uncharacterized gene in SARS-CoV-2 and the origins of the COVID-19 pandemic Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with the origins of pandemics, but are still widely overlooked. We identify ORF3c, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not more closely related pangolin-CoVs (Guangdong) or bat-CoVs (RaTG13 and RmYN02). We then document evidence of translation from ribosome profiling and conduct an evolutionary analysis at three levels: between-species (n=21 betacoronavirus genomes), between-host (n=3,978 SARS-CoV-2 consensus sequences), and within-host (401 deeply sequenced SARS-CoV-2 samples). ORF3c has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as ORF3b, an unrelated gene in other SARS-related betacoronaviruses, leading to confusion and unfounded functional inferences. Our results liken ORF3c to other viral accessory genes and stress the importance of studying OLGs.

positive: Computational Inference of Selection Underlying the Evolution of the Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection. IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.

positive: A phylodynamic workflow to rapidly gain insights into the dispersal history and dynamics of SARS-CoV-2 lineages Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of the causative virus (SARS-CoV-2) have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analysed with gold-standard phylogeographic approaches. We here describe and apply an analytical pipeline that is a compromise between fast and rigorous analytical steps. As a proof of concept, we focus on the Belgium epidemic, with one of the highest spatial density of available SARS-CoV-2 genomes. At the global scale, our analyses confirm the importance of external introduction events in establishing multiple transmission chains in the country. At the country scale, our spatially-explicit phylogeographic analyses highlight that the national lockdown had a relatively low impact on both the lineage dispersal velocity and the long-distance dispersal events within Belgium. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

positive: Sars-CoV-2 Envelope and Membrane Proteins: Structural Differences Linked to Virus Characteristics? The Coronavirus Disease 2019 (COVID-19) is a new viral infection caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2). Genomic analyses have revealed that SARS-CoV-2 is related to Pangolin and Bat coronaviruses. In this report, a structural comparison between the Sars-CoV-2 Envelope and Membrane proteins from different human isolates with homologous proteins from closely related viruses is described. The analyses here reported show the high structural similarity of Envelope and Membrane proteins to the counterparts from Pangolin and Bat coronavirus isolates. However, the comparisons have also highlighted structural differences specific of Sars-CoV-2 proteins which may be correlated to the cross-species transmission and/or to the properties of the virus. Structural modelling has been applied to map the variant sites onto the predicted three-dimensional structure of the Envelope and Membrane proteins.

positive: Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis of SARS-CoV-2-Related Viruses A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species.

positive: The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade Abstract In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.

positive: Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2 To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.

positive: Understanding evolution of SARS‐CoV‐2: a perspective from analysis of genetic diversity of RdRp gene BACKGROUND & OBJECTIVES: COVID‐19 emerged as the first example of “Disease X”, a hypothetical disease of humans caused by an unknown infectious agent that was named as novel coronavirus and subsequently designated as SARS‐CoV‐2. The origin of the outbreak at the animal market in Wuhan, China implies it as a case of zoonotic spillover. The study was designed to understand evolution of Betacoronaviruses and in particular diversification of SARS‐CoV‐2 using RdRp gene, a stable genetic marker. METHODS: Phylogenetic and population stratification analyses were carried out using Maximum likelihood and Bayesian methods, respectively. RESULTS: Molecular phylogeny using RdRp, showed that SARS‐CoV‐2 isolates cluster together. Bat‐CoV isolate RaTG13 and Pangolin‐CoVs are observed to branch off prior to SARS‐CoV‐2 cluster. While SARS‐CoV form a single cluster, Bat‐CoVs form multiple clusters. Population‐based analyses revealed that both SARS‐CoV‐2 and SARS‐CoV form separate clusters with no admixture. Bat‐CoVs were found to have single and mixed ancestry and clustered as four sub‐populations. CONCLUSIONS: Population‐based analyses of Betacoronaviruses using RdRp, revealed that SARS‐CoV‐2 is a homogeneous population. SARS‐CoV‐2 appears to have evolved from Bat‐CoV isolate RaTG13, which diversified from a common ancestor from which Pangolin‐CoVs have also evolved. The admixed Bat‐CoV sub‐populations indicate that bats serve as reservoirs harboring virus ensembles that are responsible for zoonotic spillovers such as SARS‐CoV and SARS‐CoV‐2. The extent of admixed isolates of Bat‐CoVs observed in population diversification studies underline the need for periodic surveillance of bats and other animal reservoirs for potential spillovers as a measure towards preparedness for emergence of zoonosis. This article is protected by copyright. All rights reserved.

positive: Global transmission network of SARS-CoV-2: from outbreak to pandemic BACKGROUND. The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is straining health systems around the world. Although the Chinese government implemented a number of severe restrictions on people’s movement in an attempt to contain its local and international spread, the virus had already reached many areas of the world in part due to its potent transmissibility and the fact that a substantial fraction of infected individuals develop little or no symptoms at all. Following its emergence, the virus started to generate sustained transmission in neighboring countries in Asia, Western Europe, Australia, Canada and the United States, and finally in South America and Africa. As the virus continues its global spread, a clear and evidence-based understanding of properties and dynamics of the global transmission network of SARS-CoV-2 is essential to design and put in place efficient and globally coordinated interventions. METHODS. We employ molecular surveillance data of SARS-CoV-2 epidemics for inference and comprehensive analysis of its global transmission network before the pandemic declaration. Our goal was to characterize the spatial-temporal transmission pathways that led to the establishment of the pandemic. We exploited a network-based approach specifically tailored to emerging outbreak settings. Specifically, it traces the accumulation of mutations in viral genomic variants via mutation trees, which are then used to infer transmission networks, revealing an up-to-date picture of the spread of SARS-CoV-2 between and within countries and geographic regions. RESULTS AND CONCLUSIONS. The analysis suggest multiple introductions of SARS-CoV-2 into the majority of world regions by means of heterogeneous transmission pathways. The transmission network is scale-free, with a few genomic variants responsible for the majority of possible transmissions. The network structure is in line with the available temporal information represented by sample collection times and suggest the expected sampling time difference of few days between potential transmission pairs. The inferred network structural properties, transmission clusters and pathways and virus introduction routes emphasize the extent of the global epidemiological linkage and demonstrate the importance of internationally coordinated public health measures.

positive: Tracing Back the Temporal Change of SARS-CoV-2 with Genomic Signatures The coronavirus disease (COVID-19) outbreak starting from China at the end of 2019 and its subsequent spread in many countries have given rise to thousands of coronavirus samples being collected and sequenced till date. To trace back the initial temporal change of SARS-CoV-2, the coronavirus implicated in COVID-19, we study the limited genomic sequences that were available within the first couple of months of its spread. These samples were collected under varying circumstances and highlight wide variations in their genomic compositions. In this paper, we explore whether these variations characterize the initial temporal change of SARS-CoV-2 sequences. We observe that n-mer distributions in the SARS-CoV-2 samples, which were collected at an earlier period of time, predict its collection timeline with approximately 78% accuracy. However, such a distinctive pattern disappears with the inclusion of samples collected at a later time. We further observe that isolation sources (e.g., oronasopharynx, saliva, feces, etc.) could not be predicted by the n-mer patterns in these sequences. Finally, the phylogenetic and protein-alignment analyses highlight interesting associations between SARS-CoV-2 and other coronaviruses.

positive: Complexity in SARS-CoV-2 genome data: Price theory of mutant isolates SARS-CoV-2 is a highly virulent and deadly RNA virus causing the Covid-19 pandemic and several deaths across the world. The pandemic is so fast that any concrete theory of sudden widespread of this disease is still not known. In this work, we studied and analyzed a large number of publicly available SARS-CoV-2 genomes across the world using the multifractal approach. The mutation events in the isolates obey the Markov process and exhibit very high mutational rates, which occur in six specific genes and highest in orf1ab gene, leading to virulent nature. f (α) analysis indicated that the isolates are highly asymmetric (left-skewed), revealing the richness of complexity and dominance by large fluctuations in genome structure organization. The values of Hq and Dq are found to be significantly large, showing heterogeneous genome structure self-organization, strong positive correlation in organizing the isolates, and quite sensitive to fluctuations in and around it. We then present multiple-isolates hosts-virus interaction models, and derived Price equation for the model. The phase plane analysis of the model showed asymptotic stability type bifurcation. The competition among the mutant isolates drives the trade-off of the dominant mutant isolates, otherwise confined to the present hosts.

positive: Detection of the Severe Acute Respiratory Syndrome‐Related Coronavirus and Alphacoronavirus in the Bat Population of Taiwan Bats have been demonstrated to be natural reservoirs of severe acute respiratory syndrome coronavirus (SARS CoV) and Middle East respiratory syndrome (MERS) CoV. Faecal samples from 248 individuals of 20 bat species were tested for partial RNA‐dependent RNA polymerase gene of CoV and 57 faecal samples from eight bat species were tested positive. The highest detection rate of 44% for Scotophilus kuhlii, followed by 30% for Rhinolophus monoceros. Significantly higher detection rates of coronaviral RNA were found in female bats and Scotophilus kuhlii roosting in palm trees. Phylogenetic analysis classified the positive samples into SARS‐related (SARSr) CoV, Scotophilus bat CoV 512 close to those from China and Philippines, and Miniopterus bat CoV 1A‐related lineages. Coronaviral RNA was also detected in bat guano from Scotophilus kuhlii and Myotis formosus flavus on the ground and had potential risk for human exposure. Diverse bat CoV with zoonotic potential could be introduced by migratory bats and maintained in the endemic bat population in Taiwan.

positive: Understanding evolution of SARS-CoV-2: A perspective from analysis of genetic diversity of RdRp gene Coronavirus disease 2019 emerged as the first example of "Disease X", a hypothetical disease of humans caused by an unknown infectious agent that was named as novel coronavirus and subsequently designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The origin of the outbreak at the animal market in Wuhan, China implies it as a case of zoonotic spillover. The study was designed to understand evolution of Betacoronaviruses and in particular diversification of SARS-CoV-2 using RNA dependent RNA polymerase (RdRp) gene, a stable genetic marker. Phylogenetic and population stratification analyses were carried out using maximum likelihood and Bayesian methods, respectively. Molecular phylogeny using RdRp showed that SARS-CoV-2 isolates cluster together. Bat-CoV isolate RaTG13 and Pangolin-CoVs are observed to branch off prior to SARS-CoV-2 cluster. While SARS-CoV form a single cluster, Bat-CoVs form multiple clusters. Population-based analyses revealed that both SARS-CoV-2 and SARS-CoV form separate clusters with no admixture. Bat-CoVs were found to have single and mixed ancestry and clustered as four sub-populations. Population-based analyses of Betacoronaviruses using RdRp revealed that SARS-CoV-2 is a homogeneous population. SARS-CoV-2 appears to have evolved from Bat-CoV isolate RaTG13, which diversified from a common ancestor from which Pangolin-CoVs have also evolved. The admixed Bat-CoV sub-populations indicate that bats serve as reservoirs harboring virus ensembles that are responsible for zoonotic spillovers such as SARS-CoV and SARS-CoV-2. The extent of admixed isolates of Bat-CoVs observed in population diversification studies underline the need for periodic surveillance of bats and other animal reservoirs for potential spillovers as a measure towards preparedness for emergence of zoonosis.

positive: [Etiology of epidemic outbreaks COVID-19 on Wuhan, Hubei province, Chinese People Republic associated with 2019-nCoV (Nidovirales, Coronaviridae, Coronavirinae, Betacoronavirus, Subgenus Sarbecovirus): lessons of SARS-CoV outbreak.] Results of analysis of phylogenetic, virological, epidemiological, ecological, clinical data of COVID-19 outbreaks in Wuhan, China (PRC) in comparison with SARS-2002 and MERS-2012 outbreaks allow to conclude: - the etiological agent of COVID-19 is coronavirus (2019-CoV), phylogenetically close to the SARS-CoV, isolated from human, and SARS-related viruses isolated from bats (SARS-related bat CoV viruses). These viruses belong to the Sarbecovirus subgenus, Betacoronavirus genus, Orthocoronavirinae subfamily, Coronaviridae family (Cornidovirinea: Nidovirales). COVID-19 is a variant of SARS-2002 and is different from MERS-2012 outbreak, which were caused by coronavirus belonged to the subgenus Merbecovirus of the same genus; - according to the results of phylogenetic analysis of 35 different betacoronaviruses, isolated from human and from wild animals in 2002-2019, the natural source of COVID-19 and SARS-CoV (2002) is bats of Rhinolophus genus (Rhinolophidae) and, probably, some species of other genera. An additional reservoir of the virus could be an intermediate animal species (snakes, civet, hedgehogs, badgers, etc.) that are infected by eating of infected bats. SARS-like coronaviruses circulated in bats in the interepidemic period (2003-2019); - seasonal coronaviruses (subgenus Duvinacovirus, Alphacoronavirus) are currently circulating (November 2019 - January 2020) in the European part of Russia, Urals, Siberia and the Far East of Russia, along with the influenza viruses A(H1N1)pdm09, A(H3N2), and Ð, as well as six other respiratory viruses (HPIV, HAdV, HRSV, HRV, HBoV, and HMPV).

positive: The ongoing COVID-19 epidemic in Minas Gerais, Brazil: insights from epidemiological data and SARS-CoV-2 whole genome sequencing. The recent emergence of a previously unknown coronavirus (SARS-CoV-2), first confirmed in the city of Wuhan in China in December 2019, has caused serious public health and economic issues due to its rapid dissemination worldwide. Although 61,888 confirmed cases had been reported in Brazil by 28 April 2020, little was known about the SARS-CoV-2 epidemic in the country. To better understand the recent epidemic in the second most populous state in southeast Brazil (Minas Gerais, MG), we looked at existing epidemiological data from 3 states and sequenced 40 complete genomes from MG cases using Nanopore. We found evidence of multiple independent introductions from outside MG, both from genome analyses and the overly dispersed distribution of reported cases and deaths. Epidemiological estimates of the reproductive number using different data sources and theoretical assumptions all suggest a reduction in transmission potential since the first reported case, but potential for sustained transmission in the near future. The estimated date of introduction in Brazil was consistent with epidemiological data from the first case of a returning-traveler from Lombardia, Italy. These findings highlight the unique reality of MGs epidemic and reinforce the need for real-time and continued genomic surveillance strategies as a way of understanding and therefore preparing against the epidemic spread of emerging viral pathogens.

positive: Genome-Wide Identification and Characterization of Point Mutations in the SARS-CoV-2 Genome OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and has been rapidly spreading worldwide. Although the causal relationship among mutations and the features of SARS-CoV-2 such as rapid transmission, pathogenicity, and tropism, remains unclear, our results of genomic mutations in SARS-CoV-2 may help to interpret the interaction between genomic characterization in SARS-CoV-2 and infectivity with the host. METHODS: A total of 4,254 genomic sequences of SARS-CoV-2 were collected from the Global Initiative on Sharing all Influenza Data (GISAID). Multiple sequence alignment for phylogenetic analysis and comparative genomic approach for mutation analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA), and an in-house program based on Perl language, respectively. RESULTS: Phylogenetic analysis of SARS-CoV-2 strains indicated that there were 3 major clades including S, V, and G, and 2 subclades (G.1 and G.2). There were 767 types of synonymous and 1,352 types of non-synonymous mutation. ORF1a, ORF1b, S, and N genes were detected at high frequency, whereas ORF7b and E genes exhibited low frequency. In the receptor-binding domain (RBD) of the S gene, 11 non-synonymous mutations were observed in the region adjacent to the angiotensin converting enzyme 2 (ACE2) binding site. CONCLUSION: It has been reported that the rapid infectivity and transmission of SARS-CoV-2 associated with host receptor affinity are derived from several mutations in its genes. Without these genetic mutations to enhance evolutionary adaptation, species recognition, host receptor affinity, and pathogenicity, it would not survive. It is expected that our results could provide an important clue in understanding the genomic characteristics of SARS-CoV-2.

positive: Molecular conservation and differential mutation on ORF3a gene in Indian SARS-CoV2 genomes Abstract A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory protein in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty-two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2's virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservation of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.

positive: Molecular evolution and phylogenetic analysis of SARS-CoV-2 and hosts ACE2 protein suggest Malayan pangolin as intermediary host An emergence of a novel coronavirus, causative agent of COVID19, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), occurred due to cross-species transmission. Coronaviruses are a large family of viruses able to infect a great number of hosts. Entrance of SARS-CoV-2 depends on the surface (S) protein interaction with host ACE2 protein and cleavage by TMPRSS2. ACE2 could be a species-specific barrier that interferes with bat-to-human coronavirus cross-species transmission. Molecular analysis supported bats as natural hosts for SARS-CoV and involved them in MERS-CoV origin. The genomic similarity between bat RaTG13 CoV strain and SARS-CoV-2 implicates bats in the origin of the new outbreak. Additionally, there is a hypothesis for the zoonotic transmission based on contact with Malayan pangolins by humans in Huanan seafood market in Wuhan, China. To investigate bats and pangolin as hosts in SARS-CoV-2 cross-species transmission, we perform an evolutionary analysis combining viral and host phylogenies and divergence of ACE2 and TMPRSS2 amino acid sequences between CoV hosts. Phylogeny showed SARS-like-CoV-2 strains that infected pangolin and bats are close to SARS-CoV-2. In contrast to TMPRSS2, pangolin ACE2 amino acid sequence has low evolutionary divergence compared with humans and is more divergent from bats. Comparing SARS-CoV with SARS-CoV-2 origins, pangolin has yet lower ACE2 evolutionary divergence with humans than civet—the main intermediary host of SARS-CoV. Thus, pangolin has become an opportune host to intermediates bat-to-human SARS-CoV-2 jump and entry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00321-1) contains supplementary material, which is available to authorized users.

positive: Nonstructural proteins NS7b and NS8 are likely to be phylogenetically associated with evolution of 2019-nCoV The seventh novel human infecting Betacoronavirus that causes pneumonia (2019 novel coronavirus, 2019-nCoV) originated in Wuhan, China. The evolutionary relationship between 2019-nCoV and the other human respiratory illness-causing coronavirus is not closely related. We sought to characterize the relationship of the translated proteins of 2019-nCoV with other species of Orthocoronavirinae. A phylogenetic tree was constructed from the genome sequences. A cluster tree was developed from the profiles retrieved from the presence and absence of homologs of ten 2019-nCoV proteins. The combined data were used to characterize the relationship of the translated proteins of 2019-nCoV to other species of Orthocoronavirinae. Our analysis reliably suggests that 2019-nCoV is most closely related to BatCoV RaTG13 and belongs to subgenus Sarbecovirus of Betacoronavirus, together with SARS coronavirus and Bat-SARS-like coronavirus. The phylogenetic profiling cluster of homolog proteins of one annotated 2019-nCoV protein against other genome sequences revealed two clades of ten 2019-nCoV proteins. Clade 1 consisted of a group of conserved proteins in Orthocoronavirinae comprising Orf1ab polyprotein, Nucleocapsid protein, Spike glycoprotein, and Membrane protein. Clade 2 comprised six proteins exclusive to Sarbecovirus and Hibecovirus. Two of six Clade 2 nonstructural proteins, NS7b and NS8, were exclusively conserved among 2019-nCoV, BetaCoV_RaTG, and BatSARS-like Cov. NS7b and NS8 have previously been shown to affect immune response signaling in the SARS-CoV experimental model. Thus, we speculated that knowledge of the functional changes in the NS7b and NS8 proteins during evolution may provide important information to explore the human infective property of 2019-nCoV.

positive: Molecular diversity of coronaviruses in bats Abstract The existence of coronaviruses in bats is unknown until the recent discovery of bat-SARS-CoV in Chinese horseshoe bats and a novel group 1 coronavirus in other bat species. Among 309 bats of 13 species captured from 20 different locations in rural areas of Hong Kong over a 16-month period, coronaviruses were amplified from anal swabs of 37 (12%) bats by RT-PCR. Phylogenetic analysis of RNA-dependent-RNA-polymerase (pol) and helicase genes revealed six novel coronaviruses from six different bat species, in addition to the two previously described coronaviruses. Among the six novel coronaviruses, four were group 1 coronaviruses (bat-CoV HKU2 from Chinese horseshoe bat, bat-CoV HKU6 from rickett's big-footed bat, bat-CoV HKU7 from greater bent-winged bat and bat-CoV HKU8 from lesser bent-winged bat) and two were group 2 coronaviruses (bat-CoV HKU4 from lesser bamboo bats and bat-CoV HKU5 from Japanese pipistrelles). An astonishing diversity of coronaviruses was observed in bats.

positive: Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes SUMMARY: Genome Detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present The Genome Detective Coronavirus Typing Tool that can accurately identify the novel severe acute respiratory syndrome (SARS) related coronavirus (SARS-CoV-2) sequences isolated in China and around the world. The tool can accept up to 2,000 sequences per submission and the analysis of a new whole genome sequence will take approximately one minute. The tool has been tested and validated with hundreds of whole genomes from ten coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for SARS-CoV-2. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines to stop the COVID-19 disease. AVAILABILITY: https://www.genomedetective.com/app/typingtool/cov SUPPLEMENTARY INFORMATION:

positive: Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could affect viral autophagy BACKGROUND: SARS-CoV-2 is a new coronavirus that has spread globally, infecting more than 150000 people, and being declared pandemic by the WHO. We provide here bio-informatic, evolutionary analysis of 351 available sequences of its genome with the aim of mapping genome structural variations and the patterns of selection. METHODS: A Maximum likelihood tree has been built and selective pressure has been investigated in order to find any mutation developed during the SARS-CoV-2 epidemic that could potentially affect clinical evolution of the infection. FINDING: We have found in more recent isolates the presence of two mutations affecting the Non-Structural Protein 6 (NSP6) and the Open Reding Frame10 (ORF 10) adjacent regions. Amino acidic change stability analysis suggests both mutations could confer lower stability of the protein structures. INTERPRETATION: One of the two mutations, likely developed within the genome during virus spread, could affect virus intracellular survival. Genome follow-up of SARS-CoV-2 spread is urgently needed in order to identify mutations that could significantly modify virus pathogenicity.

positive: Virus strain of a mild COVID-19 patient in Hangzhou representing a new trend in SARS-CoV-2 evolution related to Furin cleavage site We found, in our 788 confirmed COVID-19 patients, the decreased rate of severe/critical type, increased liver/kidney damage and prolonged period of nuclear acid positivity during virus dissemination, when compared with Wuhan. To investigate the underlining mechanism, we isolated one strain of SARS- CoV-2 (ZJ01) in mild COVID-19 patient and found the existence of 35 specific gene mutation by gene alignment. Further phylogenetic analysis and RSCU heat map results suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 strains of viruses worldwide (C/T type) based on the base (C or T) at positions 8824 and 28247. ZJ01 were both T at these two sites, becoming the only TT type currently identified in the world. The prediction of Furin cleavage site (FCS) and the sequence alignment of virus family indicated that FCS may be an important site of coronavirus evolution. ZJ01 had mutations near FCS (F1-2), which caused changes in the structure and the electrostatic distribution of the S protein surface, further affecting the binding capacity of Furin. Single cell sequencing and ACE2-Furin co-expression results confirmed that Furin level was higher in the whole body, especially in glands, liver, kidney and colon while FCS may help SARS-CoV-2 infect these organs. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 (the source of FCS sequence-PRRA) caused influenza, further showing potential in differentiating into mild COVID-19 subtypes.

positive: Emerging genetic diversity among clinical isolates of SARS-CoV-2: Lessons for today Abstract Considering the current pandemic of COVID-19, it is imperative to gauge the role of molecular divergence in SARS-CoV-2 with time, due to clinical and epidemiological concerns. Our analyses involving molecular phylogenetics is a step toward understanding the transmission clusters that can be correlated to pathophysiology of the disease to gain insight into virulence mechanism. As the infections are increasing rapidly, more divergence is expected followed possibly by viral adaptation. We could identify mutational hotspots which appear to be major drivers of diversity among strains, with RBD of spike protein emerging as the key region involved in interaction with ACE2 and consequently a major determinant of infection outcome. We believe that such molecular analyses correlated with clinical characteristics and host predisposition need to be evaluated at the earliest to understand viral adaptability, disease prognosis, and transmission dynamics.

positive: Genome based Evolutionary study of SARS-CoV-2 towards the Prediction of Epitope Based Chimeric Vaccine SARS-CoV-2 is known to infect the neurological, respiratory, enteric, and hepatic systems of human and has already become an unprecedented threat to global healthcare system. COVID-19, the most serious public condition caused by SARS-CoV-2 leads the world to an uncertainty alongside thousands of regular death scenes. Unavailability of specific therapeutics or approved vaccine has made the recovery of COVI-19 more troublesome and challenging. The present in silico study aimed to predict a novel chimeric vaccines by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein (S), membrane protein (M), envelope protein and nucleocapsid protein (N) were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins (S, E, M and N) reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and Normal Mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. The development of preventive measures to combat COVID-19 infections might be aided the present study. However, the in vivo and in vitro validation might be ensured with wet lab trials using model animals for the implementation of the presented data.

positive: Pros and cons of the application of evolutionary theories to the evolution of SARS-CoV-2 The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe damage to the world. With the support of classic evolutionary theories and population genetics principles, many studies on the origin of SARS-CoV-2 have revealed encouraging results but meanwhile some are still under debate. We are concerned with the validity of applying classic evolutionary theories and formula to the evolution of RNA viruses. We have raised several confounding factors like the RNA replication feature and the RNA modification systems of the hosts, which might jeopardize the validity of the application of classic methods to analyze the SARS-CoV-2 data.

positive: GENOMIC CHARACTERISATION AND PHYLOGENETIC ANALYSIS OF SARS-COV-2 IN ITALY This report describes the isolation, the molecular characterization and the phylogenetic analysis of the first three complete genomes of SARS-CoV-2 isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.

positive: Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity Monitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a mutation that leads to weaker receptor binding capability, which concerns a SARS-CoV-2 sample collected on 27th January 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and raises the alarm that the ongoing vaccine development may become futile in future epidemic if more mutations were identified. Highlights Based on the currently available genome sequence data, we proved that SARS-COV-2 genome has a much lower mutation rate and genetic diversity than SARS during the 2002-2003 outbreak. The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human. We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a sample collected on 27th January 2020.

positive: Spiking Pandemic Potential: Structural and Immunological aspects of SARS-CoV-2 SUMMARY SARS-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), an infectious respiratory disease causing thousands of deaths and overwhelming public health systems. The international spread of SARS-CoV-2 is associated with the ease of global travel, and societal dynamics, immunologic naiveté of the host population, and muted innate immune responses. Based on these factors and the expanding geographic scale of the disease, the WHO declared the COVID-19 outbreak a pandemic–the first caused by a coronavirus. In this review, we summarize the current epidemiological status of COVID-19 and consider the virological and immunological lessons, animal models, and tools developed in response to prior SARS-CoV and MERS-CoV outbreaks that can serve as resources for development of SARS-CoV-2 therapeutics and vaccines. In particular, we discuss structural insights into the SARS-CoV-2 spike protein, a major determinant of transmissibility, and discuss key molecular aspects that will aid in understanding and fighting this new global threat.

positive: SweetOrigins: Extracting Evolutionary Information from Glycans Glycans, the most diverse biopolymer and crucial for many biological processes, are shaped by evolutionary pressures stemming in particular from host-pathogen interactions. While this positions glycans as being essential for understanding and targeting host-pathogen interactions, their considerable diversity and a lack of methods has hitherto stymied progress in leveraging their predictive potential. Here, we utilize a curated dataset of 12,674 glycans from 1,726 species to develop and apply machine learning methods to extract evolutionary information from glycans. Our deep learning-based language model SweetOrigins provides evolution-informed glycan representations that we utilize to discover and investigate motifs used for molecular mimicry-mediated immune evasion by commensals and pathogens. Novel glycan alignment methods enable us to identify and contextualize virulence-determining motifs in the capsular polysaccharide of Staphylococcus aureus and Acinetobacter baumannii. Further, we show that glycan-based phylogenetic trees contain most of the information present in traditional 16S rRNA-based phylogenies and improve on the differentiation of genetically closely related but phenotypically divergent species, such as Bacillus cereus and Bacillus anthracis. Leveraging the evolutionary information inherent in glycans with machine learning methodology is poised to provide further – critically needed – insights into host-pathogen interactions, sequence-to-function relationships, and the major influence of glycans on phenotypic plasticity.

positive: Comparative analyses of SAR-CoV2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis The ongoing pandemic of the coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). We have performed an integrated sequence-based analysis of SARS-CoV2 genomes from different geographical locations in order to identify its unique features absent in SARS-CoV and other related coronavirus family genomes, conferring unique infection, facilitation of transmission, virulence and immunogenic features to the virus. The phylogeny of the genomes yields some interesting results. Systematic gene level mutational analysis of the genomes has enabled us to identify several unique features of the SARS-CoV2 genome, which includes a unique mutation in the spike surface glycoprotein (A930V (24351C>T)) in the Indian SARS-CoV2, absent in other strains studied here. We have also predicted the impact of the mutations in the spike glycoprotein function and stability, using computational approach. To gain further insights into host responses to viral infection, we predict that antiviral host-miRNAs may be controlling the viral pathogenesis. Our analysis reveals nine host miRNAs which can potentially target SARS-CoV2 genes. Interestingly, the nine miRNAs do not have targets in SARS and MERS genomes. Also, hsa-miR-27b is the only unique miRNA which has a target gene in the Indian SARS-CoV2 genome. We also predicted immune epitopes in the genomes

positive: Phylogenetic pattern of SARS-CoV-2 from COVID-19 patients from Bosnia and Herzegovina: lessons learned to optimize future molecular and epidemiological approaches Whole Genome Sequence of four samples from COVID-19 outbreaks was done in two laboratories in Bosnia and Herzegovina (Veterinary Faculty Sarajevo and Alea Genetic Center). All four BiH sequences cluster mainly with European ones (Italy, Austria, France, Sweden, Cyprus, England). The constructed phylogenetic tree indicates probable multiple independent introduction events. The success of future containment measures concernig new introductions will be highly challenging for country due to the significant proportion of BH population living abroad.

positive: Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could affect viral autophagy Abstract Background SARS-CoV-2 is a new coronavirus that has spread globally, infecting more than 150000 people, and being declared pandemic by the WHO. We provide here bio-informatic, evolutionary analysis of 351 available sequences of its genome with the aim of mapping genome structural variations and the patterns of selection. Methods A Maximum likelihood tree has been built and selective pressure has been investigated in order to find any mutation developed during the SARS-CoV-2 epidemic that could potentially affect clinical evolution of the infection. Finding We have found in more recent isolates the presence of two mutations affecting the Non-Structural Protein 6 (NSP6) and the Open Reding Frame10 (ORF 10) adjacent regions. Amino acidic change stability analysis suggests both mutations could confer lower stability of the protein structures. Interpretation One of the two mutations, likely developed within the genome during virus spread, could affect virus intracellular survival. Genome follow-up of SARS-CoV-2 spread is urgently needed in order to identify mutations that could significantly modify virus pathogenicity.

positive: Identification of super-transmitters of SARS-CoV-2 A newly emerged coronavirus, SARS-CoV-2, caused severe outbreaks of pneumonia in China in December 2019 and has since spread to various countries around the world. To probe the origin and transmission dynamics of this virus, we performed phylodynamic analysis of 247 high quality genomic sequences of viruses available in the GISAID platform as of March 05, 2020. A substantial number of earliest sequences reported in Wuhan in December 2019, including those of viruses recovered from the Huanan Seafood Market (HNSM), the site of the initial outbreak, were genetically diverse, suggesting that viruses of multiple sources were involved in the original outbreak. The viruses were subsequently disseminated to different parts of China and other countries, with diverse mutational profiles being recorded in strains recovered subsequently. Interestingly, four genetic clusters defined as Super-transmitters (STs) were found to become dominant and were responsible for the major outbreaks in various countries. Among the four clusters, ST1 is widely disseminated in Asia and the US and mainly responsible for outbreaks in the states of Washington and California in the US as well as those in South Korea at the end of February and early March, whereas ST4 contributed to the pandemic in Europe. Each ST cluster carried a signature mutation profile which allowed us to trace the origin and transmission patterns of specific viruses in different parts of the world. Using the signature mutations as markers of STs, we further analysed 1539 genome sequences reported after February 29, 2020. We found that around 90% of these genomes belonged to STs with ST4 being the dominant one and their contribution to pandemic in different continents were also depicted. The identification of these super-transmitters provides insight into the control of further transmission of SARS-CoV-2.

positive: Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection Coronavirus (CoV) disease 2019 (COVID‐19) is an ongoing pandemic caused by severe acute respiratory syndrome CoV 2 (SARS‐CoV‐2). The highly contagious SARS‐CoV‐2 belongs to the genus Betacoronavirus, and it is phylogenetically closely related to SARS‐CoV, a human CoV that caused an outbreak back in 2002 to 2003. Both SARS‐CoV‐2 and SARS‐CoV enter human cells via the interactions between viral crown‐like spike protein and human angiotensin‐converting enzyme 2 (ACE2) receptor. Here, we aim to review the involvement of ACE2 in human CoV infections by discussing the roles of ACE2 in CoV evolution, cross‐species transmissibility, and COVID‐19 susceptibility. We also provide our perspectives on COVID‐19 treatment and prevention.

positive: Controlling the SARS-CoV-2 outbreak, insights from large scale whole genome sequences generated across the world Background SARS-CoV-2 most likely evolved from a bat beta-coronavirus and started infecting humans in December 2019. Since then it has rapidly infected people around the world, with more than 4.5 million confirmed cases by the middle of May 2020. Early genome sequencing of the virus has enabled the development of molecular diagnostics and the commencement of therapy and vaccine development. The analysis of the early sequences showed relatively few evolutionary selection pressures. However, with the rapid worldwide expansion into diverse human populations, significant genetic variations are becoming increasingly likely. The current limitations on social movement between countries also offers the opportunity for these viral variants to become distinct strains with potential implications for diagnostics, therapies and vaccines. Methods We used the current sequencing archives (NCBI and GISAID) to investigate 15,487 whole genomes, looking for evidence of strain diversification and selective pressure. Results We used 6,294 SNPs to build a phylogenetic tree of SARS-CoV-2 diversity and noted strong evidence for the existence of two major clades and six sub-clades, unevenly distributed across the world. We also noted that convergent evolution has potentially occurred across several locations in the genome, showing selection pressures, including on the spike glycoprotein where we noted a potentially critical mutation that could affect its binding to the ACE2 receptor. We also report on mutations that could prevent current molecular diagnostics from detecting some of the sub-clades. Conclusion The worldwide whole genome sequencing effort is revealing the challenge of developing SARS-CoV-2 containment tools suitable for everyone and the need for data to be continually evaluated to ensure accuracy in outbreak estimations.

positive: The 2019‐new coronavirus epidemic: Evidence for virus evolution There is a worldwide concern about the new coronavirus 2019‐nCoV as a global public health threat. In this article, we provide a preliminary evolutionary and molecular epidemiological analysis of this new virus. A phylogenetic tree has been built using the 15 available whole genome sequences of 2019‐nCoV, 12 whole genome sequences of 2019‐nCoV, and 12 highly similar whole genome sequences available in gene bank (five from the severe acute respiratory syndrome, two from Middle East respiratory syndrome, and five from bat SARS‐like coronavirus). Fast unconstrained Bayesian approximation analysis shows that the nucleocapsid and the spike glycoprotein have some sites under positive pressure, whereas homology modeling revealed some molecular and structural differences between the viruses. The phylogenetic tree showed that 2019‐nCoV significantly clustered with bat SARS‐like coronavirus sequence isolated in 2015, whereas structural analysis revealed mutation in Spike Glycoprotein and nucleocapsid protein. From these results, the new 2019‐nCoV is distinct from SARS virus, probably trasmitted from bats after mutation conferring ability to infect humans.

positive: The 2019-new coronavirus epidemic: evidence for virus evolution There is concern about a new coronavirus, the 2019-nCoV, as a global public health threat. In this article, we provide a preliminary evolutionary and molecular epidemiological analysis of this new virus. A phylogenetic tree has been built using the 15 available whole genome sequence of 2019-nCoV and 12 whole genome sequences highly similar sequences available in gene bank (5 from SARS, 2 from MERS and 5 from Bat SARS-like Coronavirus). FUBAR analysis shows that the Nucleocapsid and the Spike Glycoprotein has some sites under positive pressure while homology modelling helped to explain some molecular and structural differences between the viruses. The phylogenetic tree showed that 2019.nCoV significantly clustered with Bat SARS-like Coronavirus sequence isolated in 2015, whereas structural analysis revealed mutation in S and nucleocapsid proteins. From these results, 2019nCoV could be considered a coronavirus distinct from SARS virus, probably transmitted from bats or another host where mutations conferred upon it the ability to infect humans.

positive: Comparative computational analysis of SARS-CoV-2 nucleocapsid protein epitopes in taxonomically related coronaviruses Abstract Several research lines are currently ongoing to address the multitude of facets of the pandemic COVID-19. In line with the One-Health concept, extending the target of the studies to the animals which humans are continuously interacting with may favor a better understanding of the SARS-CoV-2 biology and pathogenetic mechanisms; thus, helping to adopt the most suitable containment measures. The last two decades have already faced severe manifestations of the coronavirus infection in both humans and animals, thus, circulating epitopes from previous outbreaks might confer partial protection from SARS-CoV-2 infections. In the present study, we provide an in-silico survey of the major nucleocapsid protein epitopes and compare them with the homologues of taxonomically-related coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Protein sequence alignment provides evidence of high sequence homology for some of the investigated proteins. Moreover, structural epitope mapping by homology modelling revealed a potential immunogenic value also for specific sequences scoring a lower identity with SARS-CoV-2 nucleocapsid proteins. These evidence provide a molecular structural rationale for a potential role in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.

positive: Evolution and variation of 2019-novel coronavirus Background The current outbreak caused by novel coronavirus (2019-nCoV) in China has become a worldwide concern. As of 28 January 2020, there were 4631 confirmed cases and 106 deaths, and 11 countries or regions were affected. Methods We downloaded the genomes of 2019-nCoVs and similar isolates from the Global Initiative on Sharing Avian Influenza Database (GISAID and nucleotide database of the National Center for Biotechnology Information (NCBI). Lasergene 7.0 and MEGA 6.0 softwares were used to calculate genetic distances of the sequences, to construct phylogenetic trees, and to align amino acid sequences. Bayesian coalescent phylogenetic analysis, implemented in the BEAST software package, was used to calculate the molecular clock related characteristics such as the nucleotide substitution rate and the most recent common ancestor (tMRCA) of 2019-nCoVs. Results An isolate numbered EPI_ISL_403928 showed different phylogenetic trees and genetic distances of the whole length genome, the coding sequences (CDS) of ployprotein (P), spike protein (S), and nucleoprotein (N) from other 2019-nCoVs. There are 22, 4, 2 variations in P, S, and N at the level of amino acid residues. The nucleotide substitution rates from high to low are 1·05 × 10−2 (nucleotide substitutions/site/year, with 95% HPD interval being 6.27 × 10−4 to 2.72 × 10−2) for N, 5.34 × 10−3 (5.10 × 10−4, 1.28 × 10−2) for S, 1.69 × 10−3 (3.94 × 10−4, 3.60 × 10−3) for P, 1.65 × 10−3 (4.47 × 10−4, 3.24 × 10−3) for the whole genome, respectively. At this nucleotide substitution rate, the most recent common ancestor (tMRCA) of 2019-nCoVs appeared about 0.253-0.594 year before the epidemic. Conclusion Our analysis suggests that at least two different viral strains of 2019-nCoV are involved in this outbreak that might occur a few months earlier before it was officially reported.

positive: Whole-Genome Sequences of the Severe Acute Respiratory Syndrome Coronavirus-2 obtained from Romanian patients between March and June of 2020 Impact of mutations on the evolution of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) are needed for ongoing global efforts to track and trace the current pandemic, in order to enact effective prevention and treatment options. SARS-Co-V-2 viral genomes were detected and sequenced from 18 Romanian patients suffering from coronavirus disease-2019. Viral Spike S glycoprotein sequences were used to generate model structures and assess the role of mutations on protein stability. We integrated the phylogenetic tree within the available European SARS-Co-V-2 genomic sequences. We further provide an epidemiological overview of the pre-existing conditions that are lethal in relevant Romanian patients. Non-synonymous mutations in the viral Spike glycoprotein relating to infectivity are constructed in models of protein structures. Continuing search to limit and treat SARS-CoV-2 benefit from our contribution in delineating the viral Spike glycoprotein mutations, as well as from assessment of their role on protein stability or complex formation with human receptor angiotensin-converting enzyme 2. Our results help implement and extend worldwide genomic surveillance of coronavirus disease-2019.

positive: Phylogenomics and bioinformatics of SARS-CoV Tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. The topology and branch lengths of the phylogenetic relationships among the family Coronaviridae, including SARS-CoV, have been estimated using the replicase polyprotein. The spike protein fragments S1 (involved in receptor-binding) and S2 (involved in membrane fusion) have been found to have different mutation rates. Fragment S1 can be further divided into two regions (S1A, which comprises approximately the first 400 nucleotides, and S1B, comprising the next 280) that also show different rates of mutation. The phylogeny presented on the basis of S1B shows that SARS-CoV is closely related to MHV (murine hepatitis virus), which is known to bind the murine receptor CEACAM1. The predicted structure, accessibility and mutation rate of the S1B region is also presented. Because anti-SARS drugs based on S2 heptads have short half-lives and are difficult to manufacture, our findings suggest that the S1B region might be of interest for anti-SARS drug discovery.

positive: Genotyping coronavirus SARS-CoV-2: methods and implications The emerging global infectious COVID-19 coronavirus disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. For understanding the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. We present an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The SNP genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. Our method serves a promising tool for monitoring and tracking the epidemic of pathogenic viruses in their gradual and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.

positive: SARS-CoV-2 spread across the Colombian-Venezuelan border Venezuela and Colombia both adopted measures of containment early in response to the COVID-19 pandemic. However, Venezuelan ongoing humanitarian crisis has decimated its health care system, and forced millions of Venezuelans to flee through its porous border with Colombia. The extensive shared border, and illegal cross-border transit through improvised trails between the two countries are major challenges for public health authorities. We report the first SARS-CoV-2 genomes from Venezuela, and present a snapshot of the SARS-CoV-2 epidemiologic landscape in the Colombian-Venezuelan border region. We sequenced and assembled viral genomes from total RNA extracted from nasopharyngeal (NP) clinical specimens using a custom reference-based analysis pipeline. Three assemblies obtained were subjected to typing using the Phylogenetic Assignment of Named Global Outbreak LINeages Pangolin tool. A total of 376 publicly available SARS-CoV-2 genomes from South America were obtained from the GISAID database to perform comparative genomic analyses. Additionally, the Wuhan-1 strain was used as reference. We found that two of the SARS-CoV-2 genomes from Venezuela belonged to the B1 lineage, and the third to the B.1.13 lineage. We observed a point mutation in the Spike protein gene (D614G substitution), previously reported to be associated with increased infectivity, in all three Venezuelan genomes. An additional three mutations (R203K/G204R substitution) were present in the nucleocapsid (N) gene of one Venezuelan genome. Conclusions: Genomic sequencing demonstrates similarity between SARS-CoV-2 lineages from Venezuela and viruses collected from patients in bordering areas in Colombia and from Brazil, consistent with cross-border transit despite administrative measures including lockdowns. The presence of mutations associated with increased infectivity in the 3 Venezuelan genomes we report and Colombian SARS-CoV-2 genomes from neighboring borders areas may pose additional challenges for control of SARS-CoV-2 spread in the complex epidemiological landscape in Latin American countries. Public health authorities should carefully follow the progress of the pandemic and its impact on displaced populations within the region.

positive: Genomic variance of the 2019-nCoV coronavirus There is rising global concern for the recently emerged novel Coronavirus (2019-nCov). Full genomic sequences have been released by the worldwide scientific community in the last few weeks in order to understand the evolutionary origin and molecular characteristics of this virus. Taking advantage of all the genomic information currently available, we constructed a phylogenetic tree including also representatives of other coronaviridae, such as Bat coronavirus (BCoV) and SARS. We confirm high sequence similarity (>99%) between all sequenced 2019-nCoVs genomes available, with the closest BCoV sequence sharing 96.2% sequence identity, confirming the notion of a zoonotic origin of 2019-nCoV. Despite the low heterogeneity of the 2019-nCoV genomes, we could identify at least two hyper-variable genomic hotspots, one of which is responsible for a Serine/Leucine variation in the viral ORF8-encoded protein. Finally, we perform a full proteomic comparison with other coronaviridae, identifying key aminoacidic differences to be considered for antiviral strategies deriving from previous anti-coronavirus approaches.

positive: Comparative genomics suggests limited variability and similar evolutionary patterns between major clades of SARS-CoV-2 Phylogenomic analysis of SARS-CoV-2 as available from publicly available repositories suggests the presence of 3 prevalent groups of viral episomes (super-clades), which are mostly associated with outbreaks in distinct geographic locations (China, USA and Europe). While levels of genomic variability between SARS-CoV-2 isolates are limited, to our knowledge, it is not clear whether the observed patterns of variability in viral super-clades reflect ongoing adaptation of SARS-CoV-2, or merely genetic drift and founder effects. Here, we analyze more than 1100 complete, high quality SARS-CoV-2 genome sequences, and provide evidence for the absence of distinct evolutionary patterns/signatures in the genomes of the currently known major clades of SARS-CoV-2. Our analyses suggest that the presence of distinct viral episomes at different geographic locations are consistent with founder effects, coupled with the rapid spread of this novel virus. We observe that while cross species adaptation of the virus is associated with hypervariability of specific protein coding regions (including the RDB domain of the spike protein), the more variable genomic regions between extant SARS-CoV-2 episomes correspond with the 3’ and 5’ UTRs, suggesting that at present viral protein coding genes should not be subjected to different adaptive evolutionary pressures in different viral strains. Although this study can not be conclusive, we believe that the evidence presented here is strongly consistent with the notion that the biased geographic distribution of SARS-CoV-2 isolates should not be associated with adaptive evolution of this novel pathogen.

positive: A territory-wide study of early COVID-19 outbreak in Hong Kong community: A clinical, epidemiological and phylogenomic investigation Background: Initial cases of coronavirus disease 2019 (COVID-19) reported in Hong Kong were mostly imported cases from Mainland China. However, most cases reported in February 2020 were local infections with unknown source, indicating local community transmissions. This study aimed to report the clinical, epidemiological and phylogenomic characteristics of the local cases of COVID-19 in our community. Methods: We extracted the demographic, clinical and epidemiological data from 50 COVID-19 patients, who accounted for 53.8% of the cases in Hong Kong by the end of February 2020. We used both Nanopore and Illumina platforms to perform whole-genome sequencing (WGS) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from these patients. Phylogenetic relatedness among these local cases and their placement in the global phylogeny were examined. The evolutionary rate and divergence time of transmission were also determined. Findings: Of these 50 patients, only three (6.0%) had visited Wuhan while 43 (86.0%) did not have recent travel records. The average interval from symptom onset to hospital admission was 8.5 days. The most common signs and symptoms on admission were cough (74.0%) and fever (58.0%). Radiographic abnormality was found in 46 (92.0%) patients. Three (6.0%) patients required ICU admission. Phylogenetic analysis concurred with epidemiological investigation that 42 (84.0%) cases could be grouped into six transmission clusters. Forty-four (88.0%) cases harboured a common mutation Orf3a G251V. Global phylogeny of SARS-CoV-2 revealed that most (88.0%) cases in Hong Kong were clustered in two subclades with the strains from other countries. The estimated time to the most recent common ancestor (tMRCA) of COVID-2019 outbreak in Hong Kong was December 24, 2019 with an evolutionary rate of 3.04x10-3 substitutions per site per year. The reproduction number value was 1.84 as of February 28, 2020 in Hong Kong. Interpretation: We provided a territory-wide overview of COVID-19 in Hong Kong, which has borders connecting to Mainland China. Transmission in closed settings especially during family and religious gatherings is a hallmark of the recently reported cases. The reproduction number value indicated an ongoing outbreak in the community. Social distancing and vigilant epidemiological control are crucial to the containment of COVID-19 transmission

positive: Nonstructural proteins NS7b and NS8 are likely to be phylogenetically associated with evolution of 2019-nCoV Abstract The seventh novel human infecting Betacoronavirus that causes pneumonia (2019 novel coronavirus, 2019-nCoV) originated in Wuhan, China. The evolutionary relationship between 2019-nCoV and the other human respiratory illness-causing coronavirus is not closely related. We sought to characterize the relationship of the translated proteins of 2019-nCoV with other species of Orthocoronavirinae. A phylogenetic tree was constructed from the genome sequences. A cluster tree was developed from the profiles retrieved from the presence and absence of homologs of ten 2019-nCoV proteins. The combined data were used to characterize the relationship of the translated proteins of 2019-nCoV to other species of Orthocoronavirinae. Our analysis reliably suggests that 2019-nCoV is most closely related to BatCoV RaTG13 and belongs to subgenus Sarbecovirus of Betacoronavirus, together with SARS coronavirus and Bat-SARS-like coronavirus. The phylogenetic profiling cluster of homolog proteins of one annotated 2019-nCoV protein against other genome sequences revealed two clades of ten 2019-nCoV proteins. Clade 1 consisted of a group of conserved proteins in Orthocoronavirinae comprising Orf1ab polyprotein, Nucleocapsid protein, Spike glycoprotein, and Membrane protein. Clade 2 comprised six proteins exclusive to Sarbecovirus and Hibecovirus. Two of six Clade 2 nonstructural proteins, NS7b and NS8, were exclusively conserved among 2019-nCoV, BetaCoV_RaTG, and BatSARS-like Cov. NS7b and NS8 have previously been shown to affect immune response signaling in the SARS-CoV experimental model. Thus, we speculated that knowledge of the functional changes in the NS7b and NS8 proteins during evolution may provide important information to explore the human infective property of 2019-nCoV.

positive: Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Causing a COVID-19 Case in Morocco Here, we report a complete genome sequence obtained for a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from a nasopharyngeal swab specimen of a Moroccan patient with coronavirus disease 2019 (COVID-19).

positive: Multiple introductions, regional spread and local differentiation during the first week of COVID-19 epidemic in Montevideo, Uruguay Background After its emergence in China in December 2019, the new coronavirus disease (COVID-19) caused by SARS-CoV-2, has rapidly spread infecting more than 3 million people worldwide. South America is among the last regions hit by COVID-19 pandemic. In Uruguay, first cases were detected on March 13 th 2020 presumably imported by travelers returning from Europe. Methods We performed whole-genome sequencing of 10 SARS-CoV-2 from patients diagnosed during the first week (March 16th to 19th) of COVID-19 outbreak in Uruguay. Then, we applied genomic epidemiology using a global dataset to reconstruct the local spatio-temporal dynamics of SARS-CoV-2. Results Our phylogeographic analysis showed three independent introductions of SARS-CoV-2 from different continents. Also, we evidenced regional circulation of viral strains originally detected in Spain. Introduction of SARS-CoV-2 in Uruguay could date back as early as Feb 20th. Identification of specific mutations showed rapid local genetic differentiation. Conclusions We evidenced early independent introductions of SARS-CoV-2 that likely occurred before first cases were detected. Our analysis set the bases for future genomic epidemiology studies to understand the dynamics of SARS-CoV-2 in Uruguay and the Latin America and the Caribbean region.

positive: The origin and underlying driving forces of the SARS-CoV-2 outbreak BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

positive: [Etiology of epidemic outbreaks COVID-19 on Wuhan, Hubei province, Chinese People Republic associated with 2019-nCoV (Nidovirales, Coronaviridae, Coronavirinae, Betacoronavirus, Subgenus Sarbecovirus): lessons of SARS-CoV outbreak.] Results of analysis of phylogenetic, virological, epidemiological, ecological, clinical data of COVID-19 outbreaks in Wuhan, China (PRC) in comparison with SARS-2002 and MERS-2012 outbreaks allow to conclude: - the etiological agent of COVID-19 is coronavirus (2019-CoV), phylogenetically close to the SARS-CoV, isolated from human, and SARS-related viruses isolated from bats (SARS-related bat CoV viruses). These viruses belong to the Sarbecovirus subgenus, Betacoronavirus genus, Orthocoronavirinae subfamily, Coronaviridae family (Cornidovirinea: Nidovirales). COVID-19 is a variant of SARS-2002 and is different from MERS-2012 outbreak, which were caused by coronavirus belonged to the subgenus Merbecovirus of the same genus; - according to the results of phylogenetic analysis of 35 different betacoronaviruses, isolated from human and from wild animals in 2002-2019, the natural source of COVID-19 and SARS-CoV (2002) is bats of Rhinolophus genus (Rhinolophidae) and, probably, some species of other genera. An additional reservoir of the virus could be an intermediate animal species (snakes, civet, hedgehogs, badgers, etc.) that are infected by eating of infected bats. SARS-like coronaviruses circulated in bats in the interepidemic period (2003-2019); - seasonal coronaviruses (subgenus Duvinacovirus, Alphacoronavirus) are currently circulating (November 2019 - January 2020) in the European part of Russia, Urals, Siberia and the Far East of Russia, along with the influenza viruses A(H1N1)pdm09, A(H3N2), and В, as well as six other respiratory viruses (HPIV, HAdV, HRSV, HRV, HBoV, and HMPV).

positive: Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: a computational biology approach The newly identified SARS-CoV-2 has now been reported from around 183 countries with more than a million confirmed human cases including more than 68000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins have gotten substantial attention recently. Spike glycoprotein is widely considered as a possible target to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment tools. In this study, 483 unique variations have been identified among the genomes including 25 non-synonymous mutations and one deletion in the spike protein of SARS-CoV-2. Among the 26 variations detected, 12 variations were located at the N-terminal domain and 6 variations at the receptor-binding domain (RBD) which might alter the interaction with receptor molecules. In addition, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on our findings, potential inhibitors can be designed and tested targeting these proposed sites of variation.

positive: Era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned Unexplained pneumonia (UP) caused by a novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) emerged in China in late December 2019 and has infected more than 9000 cases by 31 January 2020. Shanghai reported the first imported case of COVID-19 (Coronavirus Disease 2019) in 20 January 2020. A combinative approach of real-time RT–PCR, CRISPR-based assay and metagenomic next-generation sequencing (mNGS) were used to diagnose this unexplained pneumonia patient. Real-time RT–PCR and CRISPR-based assay both reported positive. This sample belonged to Betacoronavirus and shared a more than 99% nucleotide (nt) identity with the Wuhan SARS-CoV-2 isolates. We further compared pros and cons of common molecular diagnostics in UP. In this study, we illustrated the importance of combining molecular diagnostics to rule out common pathogens and performed mNGS to obtain unbiased potential pathogen result for the diagnosis of UP.

positive: Full-genome sequences of the first two SARS-CoV-2 viruses from India BACKGROUND & OBJECTIVES: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. METHODS: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. RESULTS: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. INTERPRETATION & CONCLUSIONS: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

positive: Characterization of SARS-CoV-2 viral diversity within and across hosts In light of the current COVID-19 pandemic, there is an urgent need to accurately infer the evolutionary and transmission history of the virus to inform real-time outbreak management, public health policies and mitigation strategies. Current phylogenetic and phylodynamic approaches typically use consensus sequences, essentially assuming the presence of a single viral strain per host. Here, we analyze 621 bulk RNA sequencing samples and 7,540 consensus sequences from COVID-19 patients, and identify multiple strains of the virus, SARS-CoV-2, in four major clades that are prevalent within and across hosts. In particular, we find evidence for (i) within-host diversity across phylogenetic clades, (ii) putative cases of recombination, multi-strain and/or superinfections as well as (iii) distinct strain profiles across geographical locations and time. Our findings and algorithms will facilitate more detailed evolutionary analyses and contact tracing that specifically account for within-host viral diversity in the ongoing COVID-19 pandemic as well as future pandemics.

positive: The Role of Phylogenetic Analysis in Clarifying the Infection Source of a COVID-19 Patient

positive: Whole Genome Comparison of Pakistani Corona Virus with Chinese and US Strains along with its Predictive Severity of COVID-19 Recently submitted 784 SARS-nCoV2 whole genome sequences from NCBI Virus database were taken for constructing phylogenetic tree to look into their similarities. Pakistani strain MT240479 (Gilgit1-Pak) was found in close proximity to MT184913 (CruiseA-USA), while the second Pakistani strain MT262993 (Manga-Pak) was neighboring to MT039887 (WI-USA) strain in the constructed cladogram in this article. Afterward, four whole genome SARS-nCoV2 strain sequences were taken for variant calling analysis, those who appeared nearest relative in the earlier cladogram constructed a week time ago. Among those two Pakistani strains each of 29,836 bases were compared against MT263429 from (WI-USA) of 29,889 bases and MT259229 (Wuhan-China) of 29,864 bases. We identified 31 variants in both Pakistani strains, (Manga-Pak vs USA=2del+7SNPs, Manga-Pak vs Chinese=2del+2SNPs, Gilgit1-Pak vs USA=10SNPs, Gilgit1-Pak vs Chinese=8SNPs), which caused alteration in ORF1ab, ORF1a and N genes with having functions of viral replication and translation, host innate immunity and viral capsid formation respectively. These novel variants are assumed to be liable for low mortality rate in Pakistan with 385 as compared to USA with 63,871 and China with 4,633 deaths by May 01, 2020. However functional effects of these variants need further confirmatory studies. Moreover, mutated N & ORF1a proteins in Pakistani strains were also analyzed by 3D structure modelling, which give another dimension of comparing these alterations at amino acid level. In a nutshell, these novel variants are assumed to be linked with reduced mortality of COVID-19 in Pakistan along with other influencing factors, these novel variants would also be useful to understand the virulence of this virus and to develop indigenous vaccines and therapeutics.

positive: Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event BACKGROUND: A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. METHODS: Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. RESULTS: Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat_SARS-like coronavirus sequences. Specifically, in the 5'-part spanning the first 11,498 nucleotides and the last 3'-part spanning 24,341-30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat_SARS-like coronavirus sequences, whereas in the middle region spanning the 3'-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. CONCLUSIONS: The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

positive: Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach The newly identified SARS-CoV-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. Spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment. In this study, 483 unique variations have been identified among the genomes of SARS-CoV-2 including 25 nonsynonymous mutations and one deletion in the spike (S) protein. Among the 26 variations detected, 12 variations were located at the N-terminal domain and 6 variations at the receptor-binding domain (RBD) which might alter the interaction of S protein with the host receptor angiotensin converting enzyme-2 (ACE2). Besides, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on results reported herein, potential inhibitors against S protein can be designed by considering these variations and their impact on protein structure.

positive: The global spread of 2019-nCoV: a molecular evolutionary analysis The global spread of the 2019-nCoV is continuing and is fast moving, as indicated by the WHO raising the risk assessment to high. In this article, we provide a preliminary phylodynamic and phylogeographic analysis of this new virus. A Maximum Clade Credibility tree has been built using the 29 available whole genome sequences of 2019-nCoV and two whole genome sequences that are highly similar sequences from Bat SARS-like Coronavirus available in GeneBank. We are able to clarify the mechanism of transmission among the countries which have provided the 2019-nCoV sequence isolates from their patients. The Bayesian phylogeographic reconstruction shows that the 2019–2020 nCoV most probably originated from the Bat SARS-like Coronavirus circulating in the Rhinolophus bat family. In agreement with epidemiological observations, the most likely geographic origin of the new outbreak was the city of Wuhan, China, where 2019-nCoV time of the most recent common ancestor emerged, according to molecular clock analysis, around November 25(th), 2019. These results, together with previously recorded epidemics, suggest a recurring pattern of periodical epizootic outbreaks due to Betacoronavirus. Moreover, our study describes the same population genetic dynamic underlying the SARS 2003 epidemic, and suggests the urgent need for the development of effective molecular surveillance strategies of Betacoronavirus among animals and Rhinolophus of the bat family.

positive: Phylodynamics of SARS-CoV-2 transmission in Spain Objectives SARS-CoV-2 whole-genome analysis has identified three large clades spreading worldwide, designated G, V and S. This study aims to analyze the diffusion of SARS-CoV-2 in Spain/Europe. Methods Maximum likelihood phylogenetic and Bayesian phylodynamic analyses have been performed to estimate the most probable temporal and geographic origin of different phylogenetic clusters and the diffusion pathways of SARS-CoV-2. Results Phylogenetic analyses of the first 28 SARS-CoV-2 whole genome sequences obtained from patients in Spain revealed that most of them are distributed in G and S clades (13 sequences in each) with the remaining two sequences branching in the V clade. Eleven of the Spanish viruses of the S clade and six of the G clade grouped in two different monophyletic clusters (S-Spain and G-Spain, respectively), with the S-Spain cluster also comprising 8 sequences from 6 other countries from Europe and the Americas. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated in the city of Wuhan, China, around November 24, 2019, with a 95% highest posterior density (HPD) interval from October 30-December 17, 2019. The origin of S-Spain and G-Spain clusters were estimated in Spain around February 14 and 18, 2020, respectively, with a possible ancestry of S-Spain in Shanghai. Conclusions Multiple SARS-CoV-2 introductions have been detected in Spain and at least two resulted in the emergence of locally transmitted clusters, with further dissemination of one of them to at least 6 other countries. These results highlight the extraordinary potential of SARS-CoV-2 for rapid and widespread geographic dissemination.

positive: Genomic variations in SARS-CoV-2 genomes from Gujarat: Underlying role of variants in disease epidemiology Humanity has seen numerous pandemics during its course of evolution. The list includes many such as measles, Ebola, SARS, MERS, etc. Latest edition to this pandemic list is COVID-19, caused by the novel coronavirus, SARS-CoV-2. As of 4th July 2020, COVID-19 has affected over 10 million people from 170+ countries, and 5,28,364 deaths. Genomic technologies have enabled us to understand the genomic constitution of the pathogens, their virulence, evolution, rate of mutations, etc. To date, more than 60,000 virus genomes have been deposited in the public depositories like GISAID and NCBI. While we are writing this, India is the 3rd most-affected country with COVID-19 with 0.6 million cases, and >18000 deaths. Gujarat is the fourth highest affected state with 5.44 percent death rate compared to national average of 2.8 percent. Here, 361 SARS-CoV-2 genomes from across Gujarat have been sequenced and analyzed in order to understand its phylogenetic distribution and variants against global and national sequences. Further, variants were analyzed from diseased and recovered patients from Gujarat and the World to understand its role in pathogenesis. From missense mutations, found from Gujarat SARS-CoV-2 genomes, C28854T, deleterious mutation in nucleocapsid (N) gene was found to be significantly associated with mortality in patients. The other significant deleterious variant found in diseased patients from Gujarat and the world is G25563T, which is located in Orf3a and has a potential role in viral pathogenesis. SARS-CoV-2 genomes from Gujarat are forming distinct cluster under GH clade of GISAID.

positive: An Analysis of SARS-CoV-2 Using ViReport The ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of cases and hundreds of thousands of deaths. Given the current lack of treatments or vaccines available, it may be useful to trace the evolu-tion and spread of the virus to better develop methods of preventative intervention. In this study, we analyzed over 4,000 full genome sequences of human SARS-CoV-2 using novel tool ViReport [13], an automated workflow for performing phylogenetic analyses on viral sequences and generating comprehensive molecular epidemiologi-cal reports. The complete ViReport output can be found at https://github.com/mirandajsong/ViReport-SARS-CoV-2.

positive: Insights into molecular evolution recombination of pandemic SARS-CoV-2 using Saudi Arabian sequences The recently emerged SARS-CoV-2 (Coronaviridae; Betacoronavirus) is the underlying cause of COVID-19 disease. Here we assessed SARS-CoV2 from the Kingdom of Saudi Arabia alongside sequences of SARS-CoV, bat SARS-like CoVs and MERS-CoV, the latter currently detected in this region. Phylogenetic analysis, natural selection investigation and genome recombination analysis were performed. Our analysis showed that all Saudi SARS-CoV-2 sequences are of the same origin and closer proximity to bat SARS-like CoVs, followed by SARS-CoVs, however quite distant to MERS-CoV. Moreover, genome recombination analysis revealed two recombination events between SARS-CoV-2 and bat SARS-like CoVs. This was further assessed by S gene recombination analysis. These recombination events may be relevant to the emergence of this novel virus. Moreover, positive selection pressure was detected between SARS-CoV-2, bat SL-CoV isolates and human SARS-CoV isolates. However, the highest positive selection occurred between SARS-CoV-2 isolates and 2 bat-SL-CoV isolates (Bat-SL-RsSHC014 and Bat-SL-CoVZC45). This further indicates that SARS-CoV-2 isolates were adaptively evolved from bat SARS-like isolates, and that a virus with originating from bats triggered this pandemic. This study thuds sheds further light on the origin of this virus. AUTHOR SUMMARY The emergence and subsequent pandemic of SARS-CoV-2 is a unique challenge to countries all over the world, including Saudi Arabia where cases of the related MERS are still being reported. Saudi SARS-CoV-2 sequences were found to be likely of the same or similar origin. In our analysis, SARS-CoV-2 were more closely related to bat SARS-like CoVs rather than to MERS-CoV (which originated in Saudi Arabia) or SARS-CoV, confirming other phylogenetic efforts on this pathogen. Recombination and positive selection analysis further suggest that bat coronaviruses may be at the origin of SARS-CoV-2 sequences. The data shown here give hints on the origin of this virus and may inform efforts on transmissibility, host adaptation and other biological aspects of this virus.

positive: Molecular immune pathogenesis and diagnosis of COVID-19 Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of SARS-CoV-2 has been marked as the third introduction of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) in the twenty-first century. In this minireview, we provide a brief introduction of the general features of SARS-CoV-2 and discuss current knowledge of molecular immune pathogenesis, diagnosis and treatment of COVID-19 on the base of the present understanding of SARS-CoV and MERS-CoV infections, which may be helpful in offering novel insights and potential therapeutic targets for combating the SARS-CoV-2 infection.

positive: Computational Inference of Selection Underlying the Evolution of the Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection.IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.

positive: Sequencing identifies multiple, early introductions of SARS-CoV2 to New York City Region Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 236 SARS-CoV2 sequences from cases in the New York City metropolitan area during the initial stages of the 2020 COVID-19 outbreak. The majority of cases throughout the region had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that the majority were most related to cases from Europe. Our data are consistent with numerous seed transmissions from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of real-time genomic surveillance in addition to traditional epidemiological indicators.

positive: A distinct phylogenetic cluster of Indian SARS-CoV-2 isolates From an isolated epidemic, COVID-19 has now emerged as a global pandemic. The availability of genomes in the public domain following the epidemic provides a unique opportunity to understand the evolution and spread of the SARS-CoV-2 virus across the globe. The availability of whole genomes from multiple states in India prompted us to analyse the phylogenetic clusters of genomes in India. We performed whole-genome sequencing for 64 genomes making a total of 361 genomes from India, followed by phylogenetic clustering, substitution analysis, and dating of the different phylogenetic clusters of viral genomes. We describe a distinct phylogenetic cluster (Clade I / A3i) of SARS-CoV-2 genomes from India, which encompasses 41% of all genomes sequenced and deposited in the public domain from multiple states in India. Globally 3.5% of genomes, which till date could not be mapped to any distinct known cluster fall in this newly defined clade. The cluster is characterized by a core set of shared genetic variants – C6312A (T2016K), C13730T (A88V/A97V), C23929T, and C28311T (P13L). Further, the cluster is also characterized by a nucleotide substitution rate of 1.4 × 10−3 variants per site per year, lower than the prevalent A2a cluster, and predominantly driven by variants in the E and N genes and relative sparing of the S gene. Epidemiological assessments suggest that the common ancestor emerged in the month of February 2020 and possibly resulted in an outbreak followed by countrywide spread, as evidenced by the low divergence of the genomes from across the country. To the best of our knowledge, this is the first comprehensive study characterizing the distinct and predominant cluster of SARS-CoV-2 in India.

positive: Integrated genomic view of SARS-CoV-2 in India India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

positive: Genomic diversity and hotspot mutations in 30,983 SARS-CoV-2 genomes: moving toward a universal vaccine for the “confined virus”? The Coronavirus disease 19 (COVID-19) pandemic has been ongoing since its onset in late November 2019 in Wuhan, China. To date, the SARS-CoV-2 virus has infected more than 8 million people worldwide and killed over 5% of them. Efforts are being made all over the world to control the spread of the disease and most importantly to develop a vaccine. Understanding the genetic evolution of the virus, its geographic characteristics and stability is particularly important for developing a universal vaccine covering all circulating strains of SARS-CoV-2 and for predicting its efficacy. In this perspective, we analyzed the sequences of 30,983 complete genomes from 80 countries located in six geographical zones (Africa, Asia, Europe, North & South America, and Oceania) isolated from December 24, 2019 to May 13, 2020, and compared them to the reference genome. Our in-depth analysis revealed the presence of 3,206 variant sites compared to the reference Wuhan-Hu-1 genome, with a distribution that is largely uniform over all continents. Remarkably, a low frequency of recurrent mutations was observed; only 182 mutations (5.67%) had a prevalence greater than 1%. Nevertheless, fourteen hotspot mutations (> 10%) were identified at different locations, seven at the ORF1ab gene (in regions coding for nsp2, nsp3, nsp6, nsp12, nsp13, nsp14 and nsp15), three in the nucleocapsid protein, one in the spike protein, one in orf3a, and one in orf8. Moreover, 35 non-synonymous mutations were identified in the receptor-binding domain (RBD) of the spike protein with a low prevalence (<1%) across all genomes, of which only four could potentially enhance the binding of the SARS-CoV-2 spike protein to the human receptor ACE2. These results along with the phylogenetic analysis demonstrate that the virus does not have a significant divergence at the protein level compared to the reference both among and within different geographical areas. Unlike the influenza virus or HIV viruses, the slow rate of mutation of SARS-CoV-2 makes the potential of developing an effective global vaccine very likely.

positive: Genomic epidemiology of a densely sampled COVID19 outbreak in China Analysis of genetic sequence data from the pandemic SARS Coronavirus 2 can provide insights into epidemic origins, worldwide dispersal, and epidemiological history. With few exceptions, genomic epidemiological analysis has focused on geographically distributed data sets with few isolates in any given location. Here we report an analysis of 20 whole SARS-CoV 2 genomes from a single relatively small and geographically constrained outbreak in Weifang, People's Republic of China. Using Bayesian model-based phylodynamic methods, we estimate the reproduction number for the outbreak to be 1.99(95% CI:1.48-3.14). We further estimate the number of infections through time and compare these estimates to confirmed diagnoses by the Weifang Centers for Disease Control. We find that these estimates are consistent with reported cases and there is unlikely to be a large undiagnosed burden of infection over the period we studied.

positive: Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins. The outbreak of COVID-19 poses unprecedent challenges to global health1. The new coronavirus, SARS-CoV-2, shares high sequence identity to SARS-CoV and a bat coronavirus RaTG132. While bats may be the reservoir host for various coronaviruses3,4, whether SARS-CoV-2 has other hosts remains ambiguous. In this study, one coronavirus isolated from a Malayan pangolin showed 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S genes, respectively. In particular, the receptor-binding domain within the S protein of the Pangolin-CoV is virtually identical to that of SARS-CoV-2, with one noncritical amino acid difference. Results of comparative genomic analysis suggest that SARS-CoV-2 might have originated from the recombination of a Pangolin-CoV-like virus with a Bat-CoV-RaTG13-like virus. The Pangolin-CoV was detected in 17 of 25 Malayan pangolins analyzed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against Pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus that is highly related to SARS-CoV-2 in pangolins suggests that they have the potential to act as the intermediate host of SARS-CoV-2. The newly identified coronavirus in the most-trafficked mammal could represent a future threat to public health if wildlife trade is not effectively controlled.

positive: Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV After the outbreak of the severe acute respiratory syndrome (SARS) in the world in 2003, human coronaviruses (HCoVs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. Recently, a new emerged HCoV isolated from the respiratory epithelium of unexplained pneumonia patients in the Wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus causes acute lung symptoms, leading to a condition that has been named as "coronavirus disease 2019" (COVID-19). The emergence of SARS-CoV-2 and of SARS-CoV caused widespread fear and concern and has threatened global health security. There are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. The goal of this work is to systematically review and compare between SARS-CoV and SARS-CoV-2 in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms.

positive: Distinct genetic spectrums and evolution patterns of SARS-CoV-2 Four signature groups of single-nucleotide variants (SNVs) were identified using two-way clustering method in about twenty thousand high quality and high coverage SARS-CoV-2 complete genome sequences. Some frequently occurred SNVs predominate but are mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguished evolution patterns overtime. Although it was rare, our data indicated possible cross-infections with multiple groups of SNVs existed simultaneously in some patients, suggesting infections from different SARS-CoV-2 clades or potential re-combination of SARS-CoV-2 sequences. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tend to occur at the sites where one bat RaTG13 coronavirus sequences differ from Wuhan-Hu-1 genome, indicating the tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.

positive: Unsupervised cluster analysis of SARS-CoV-2 genomes reflects its geographic progression and identifies distinct genetic subgroups of SARS-CoV-2 virus Over 10,000 viral genome sequences of the SARS-CoV-2 virus have been made readily available during the ongoing coronavirus pandemic since the initial genome sequence of the virus was released on the open access Virological website (http://virological.org/) early on January 11. We utilize the published data on the single stranded RNAs of 11, 132 SARS-CoV-2 patients in the GISAID (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) database, which contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Among many important research questions which are currently being investigated, one aspect pertains to the genetic characterization/classification of the virus. We analyze data on the nucleotide sequencing of the virus and geographic information of a subset of 7, 640 SARS-CoV-2 patients without missing entries that are available in the GISAID database. Instead of modelling the mutation rate, applying phylogenetic tree approaches, etc., we here utilize a model-free clustering approach that compares the viruses at a genome-wide level. We apply principal component analysis to a similarity matrix that compares all pairs of these SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index (Jaccard, 1901; Tan et al., 2005; Prokopenko et al., 2016; Schlauch et al., 2017). Our analysis results of the SARS-CoV-2 genome data illustrates the geographic and chronological progression of the virus, starting from the first cases that were observed in China to the current wave of cases in Europe and North America. We also observe that, based on their sequence data, the SARS-CoV-2 viruses cluster in distinct genetic subgroups. It is the subject of ongoing research to examine whether the genetic subgroup could be related to diseases outcome and its potential implications for vaccine development.

positive: Recombination and purifying selection preserves covariant movements of mosaic SARS-CoV-2 protein S In depth evolutionary and structural analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from bats, pangolins, and humans are necessary to assess the role of natural selection and recombination in the emergence of the current pandemic strain. The SARS-CoV-2 S glycoprotein unique features have been associated with efficient viral spread in the human population. Phylogeny-based and genetic algorithm methods clearly show that recombination events between viral progenitors infecting animal hosts led to a mosaic structure in the S gene. We identified recombination coldspots in the S glycoprotein and strong purifying selection. Moreover, although there is little evidence of diversifying positive selection during host-switching, structural analysis suggests that some of the residues emerged along the ancestral lineage of current pandemic strains may contribute to enhanced ability to infect human cells. Interestingly, recombination did not affect the long-range covariant movements of SARS-CoV-2 S glycoprotein monomer in pre-fusion conformation but, on the contrary, could contribute to the observed overall viral efficiency. Our dynamic simulations revealed that the movements between the host cell receptor binding domain (RBD) and the novel furin-like cleavage site are correlated. We identified threonine 333 (under purifying selection), at the beginning of the RBD, as the hinge of the opening/closing mechanism of the SARS-CoV-2 S glycoprotein monomer functional to hACE2 binding. Our findings support a scenario where ancestral recombination and fixation of amino acid residues in the RBD of the S glycoprotein generated a virus with unique features, capable of extremely efficient infection of the human host.

positive: From SARS-CoV to SARS-CoV-2: safety and broad-spectrum are important for coronavirus vaccine development The global pandemic of COVID-19 caused by SARS-CoV-2 (also known as 2019-nCoV and HCoV-19) has posed serious threats to public health and economic stability worldwide, thus calling for development of vaccines against SARS-CoV-2 and other emerging and reemerging coronaviruses. Since SARS-CoV-2 and SARS-CoV have high similarity of their genomic sequences and share the same cellular receptor (ACE2), it is essential to learn the lessons and experiences from the development of SARS-CoV vaccines for the development of SARS-CoV-2 vaccines. In this review, we summarized the current knowledge on the advantages and disadvantages of the SARS-CoV vaccine candidates and prospected the strategies for the development of safe, effective and broad-spectrum coronavirus vaccines for prevention of infection by currently circulating SARS-CoV-2 and other emerging and reemerging coronaviruses that may cause future epidemics or pandemics.

positive: Analysis of Possible Intermediate Hosts of the New Coronavirus SARS-CoV-2

positive: Molecular Advances in Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information.

positive: Extensive diversity of coronaviruses in bats from China Abstract To help reveal the diversity and evolution of bat coronaviruses we collected 1067 bats from 21 species in China. A total of 73 coronaviruses (32 alphacoronaviruses and 41 betacoronaviruses) were identified in these bats, with an overall prevalence of 6.84%. All newly-identified betacoronaviruses were SARS-related Rhinolophus bat coronaviruses (SARSr-Rh-BatCoV). Importantly, with the exception of the S gene, the genome sequences of the SARSr-Rh-BatCoVs sampled in Guizhou province were closely related to SARS-related human coronavirus. Additionally, the newly-identified alphacoronaviruses exhibited high genetic diversity and some may represent novel species. Our phylogenetic analyses also provided insights into the transmission of these viruses among bat species, revealing a general clustering by geographic location rather than by bat species. Inter-species transmission among bats from the same genus was also commonplace in both the alphacoronaviruses and betacoronaviruses. Overall, these data suggest that high contact rates among specific bat species enable the acquisition and spread of coronaviruses.

positive: Insights into Cross-species Evolution of Novel Human Coronavirus 2019-nCoV and Defining Immune Determinants for Vaccine Development Novel Coronavirus (nCoV) outbreak in the city of Wuhan, China during December 2019, has now spread to various countries across the globe triggering a heightened containment effort. This human pathogen is a member of betacoronavirus genus carrying 30 kilobase of single positive-sense RNA genome. Understanding the evolution, zoonotic transmission, and source of this novel virus would help accelerating containment and prevention efforts. The present study reported detailed analysis of 2019-nCoV genome evolution and potential candidate peptides for vaccine development. This nCoV genotype might have been evolved from a bat-CoV by accumulating non-synonymous mutations, indels, and recombination events. Structural proteins Spike (S), and Membrane (M) had extensive mutational changes, whereas Envelope (E) and Nucleocapsid (N) proteins were very conserved suggesting differential selection pressures exerted on 2019-nCoV during evolution. Interestingly, 2019-nCoV Spike protein contains a 39 nucleotide (5’-aAT GGT GTT GAA GGT TTT AAT TGT TAC TTT CCT TTA CAA Tca-3’) sequence insertion, which shares homology to fish genomic sequence of Myripristis murdjan, an abundant fish type in Indo-Pacific Ocean. Furthermore, we identified eight high binding affinity (HBA) CD4 T-cell epitopes in the S, E, M and N proteins, which can be commonly recognized by HLA-DR alleles of Asia and Asia-Pacific Region population. These immunodominant epitopes can be incorporated in universal subunit CoV vaccine. Diverse HLA types and variations in the epitope binding affinity may contribute to the wide range of immunopathological outcomes of circulating virus in humans. Our findings emphasize the requirement for continuous surveillance of CoV strains in live animal markets to better understand the viral adaptation to human host and to develop practical solutions to prevent the emergence of novel pathogenic CoV strains.

positive: Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here we use two different sampling methods to monitor SARS-CoV-2 in wastewater over a 17-day period and sequencing is used to infer viral ancestry. While SARS-CoV-2 is detected over the entire time course, viral RNA has been steadily decreasing over the last week, suggesting that state mandated social isolation is having a measurable impact on containment of the outbreak.

positive: Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

positive: SARS-CoV-2: virus mutations in specific European populations Abstract The SARS-CoV-2 virus is being intensively studied, particularly, its evolution in the increasingly available sequences between countries/continents with classical phylogenetic tree representation. More recently, certain protein mutations are correlated with specific functional impacts. Our clinical data from patients suggest different clinical symptoms between European countries. Among others, SARS-CoV-2 mutations could explain these disparities. Our analyses point out an association of diverse mutations, including co-evolving ones, in a few SARS-CoV-2 proteins, with specific countries. We therefore suggest combining clinical information from patients and the determination of the associated SARS-CoV-2 genome to better understand the specific symptoms.

positive: Comprehensive genome analysis of 6,000 USA SARS-CoV-2 isolates reveals haplotype signatures and localized transmission patterns by state and by country Genomic analysis of SARS-CoV-2 sequences is crucial in determining the effectiveness of prudent safer at home measures in the United States (US). By haplotype analysis of 6,356 US isolates, we identified a pattern of strongly localized outbreaks at the city-, state-, and country-levels, and temporal transmissions. This points to the effectiveness of existing travel restriction policies and public health measures in controlling the transmission of SARS-CoV-2.

positive: Comparative genomics provides an operational classification system and reveals early emergence and biased spatio-temporal distribution of SARS-CoV-2 Effective systems for the analysis of molecular data are of fundamental importance for real-time monitoring of the spread of infectious diseases and the study of pathogen evolution. While the Nextstrain and GISAID portals offer widely used systems for the classification of SARS-CoV-2 genomes, both present relevant limitations. Here we propose a highly reproducible method for the systematic classification of SARS-CoV-2 viral types. To demonstrate the validity of our approach, we conduct an extensive comparative genomic analysis of more than 20,000 SARS-CoV-2 genomes. Our classification system delineates 12 clusters and 4 super-clusters in SARS-CoV-2, with a highly biased spatio-temporal distribution worldwide, and provides important observations concerning the evolutionary processes associated with the emergence of novel viral types. Based on the estimates of SARS-CoV-2 evolutionary rate and genetic distances of genomes of the early pandemic phase, we infer that SARS-CoV-2 could have been circulating in humans since August-November 2019. The observed pattern of genomic variability is remarkably similar between all clusters and super-clusters, being UTRs and the s2m element, a highly conserved secondary structure element, the most variable genomic regions. While several polymorphic sites that are specific to one or more clusters were predicted to be under positive or negative selection, overall, our analyses also suggest that the emergence of novel genome types is unlikely to be driven by widespread convergent evolution and independent fixation of advantageous substitutions. While, in the absence of rigorous experimental validation, several questions concerning the evolutionary processes and the phenotypic characteristics (increased/decreased virulence) remain open, we believe that the approach outlined in this study can be of relevance for the tracking and functional characterization of different types of SARS-CoV-2 genomes.

positive: Importation of SARS-CoV-2 infection leads to major COVID-19 epidemic in Taiwan Abstract Objective COVID-19 has recently become a pandemic affecting many countries worldwide. This study aims to evaluate current status of COVID-19 in Taiwan and analyze the source of infection. Methods National data regarding SARS-CoV-2 infection were obtained from Taiwan CDC at the end of April, 2020. These data were subjected for analysis of the current status and correlation between indigenous and imported COVID-19 cases. Phylogenetic tree was performed to analyze the phylogeny of Taiwanese SARS-CoV-2 isolates. Results The initial case of SARS-CoV-2 infection in Taiwan was detected on January 21, 2020. Epidemiological data indicate that by April 30, there were a total of 429 COVID-19 confirmed cases with the death rate of 1.3%. Most of cases were identified as imported (79.9%; 343/429) with majority transmitted from United States of America (22.1%) and United Kingdom (17.6%). Results from phylogenetic tree analyses indicate that the Taiwanese SARS-CoV-2 isolates were clustered with the SARS-CoV-2 isolates from other countries (bootstrap value 98%) and sub-clustered with bat SARS-like coronaviruses (bootstrap value 99%). Conclusion This study suggests that importation of SARS-CoV-2 infection was the primary risk-factor resulting in the COVID-19 epidemic in Taiwan.

positive: Origin of imported SARS-CoV-2 strains in The Gambia identified from Whole Genome Sequences Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a positive-sense single stranded RNA virus with high human transmissibility. This study generated Whole Genome data to determine the origin and pattern of transmission of SARS-CoV-2 from the first six cases tested in The Gambia. Total RNA from SARS-CoV-2 was extracted from inactivated nasopharyngeal-oropharyngeal swabs of six cases and converted to cDNA following the ARTIC COVID-19 sequencing protocol. Libraries were constructed with the NEBNext ultra II DNA library prep kit for Illumina and Oxford Nanopore Ligation sequencing kit and sequenced on Illumina MiSeq and Nanopore GridION, respectively. Sequencing reads were mapped to the Wuhan reference genome and compared to eleven other SARS-CoV-2 strains of Asian, European and American origins. A phylogenetic tree was constructed with the consensus genomes for local and non-African strains. Three of the Gambian strains had a European origin (UK and Spain), two strains were of Asian origin (Japan). In The Gambia, Nanopore and Illumina sequencers were successfully used to identify the sources of SARS-CoV-2 infection in COVID-19 cases.

positive: Positive selection of ORF3a and ORF8 genes drives the evolution of SARS-CoV-2 during the 2020 COVID-19 pandemic In this study, we analyzed full-length SARS-CoV-2 genomes from multiple countries to determine early trends in the evolutionary dynamics of the novel COVID-19 pandemic. Results indicated SARS-CoV-2 evolved early into at least three phylogenetic groups, characterized by positive selection at specific residues of the accessory proteins OFR3a and ORF8a. We also report evidence of epistatic interactions among sites in the genome that may be important in the generation of variants adapted to humans. These observations might impact not only public health, but also suggest more studies are needed to understand the genetic mechanisms that may affect the development of therapeutic and preventive tools, like antivirals and vaccines.

positive: Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study Summary Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported from China in January, 2020. SARS-CoV-2 is efficiently transmitted from person to person and, in 2 months, has caused more than 82 000 laboratory-confirmed cases of coronavirus disease 2019 (COVID-19) and 2800 deaths in 46 countries. The total number of cases and deaths has surpassed that of the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV). Although both COVID-19 and severe acute respiratory syndrome (SARS) manifest as pneumonia, COVID-19 is associated with apparently more efficient transmission, fewer cases of diarrhoea, increased mental confusion, and a lower crude fatality rate. However, the underlying virus–host interactive characteristics conferring these observations on transmissibility and clinical manifestations of COVID-19 remain unknown. Methods We systematically investigated the cellular susceptibility, species tropism, replication kinetics, and cell damage of SARS-CoV-2 and compared findings with those for SARS-CoV. We compared SARS-CoV-2 and SARS-CoV replication in different cell lines with one-way ANOVA. For the area under the curve comparison between SARS-CoV-2 and SARS-CoV replication in Calu3 (pulmonary) and Caco2 (intestinal) cells, we used Student's t test. We analysed cell damage induced by SARS-CoV-2 and SARS-CoV with one-way ANOVA. Findings SARS-CoV-2 infected and replicated to comparable levels in human Caco2 cells and Calu3 cells over a period of 120 h (p=0·52). By contrast, SARS-CoV infected and replicated more efficiently in Caco2 cells than in Calu3 cells under the same multiplicity of infection (p=0·0098). SARS-CoV-2, but not SARS-CoV, replicated modestly in U251 (neuronal) cells (p=0·036). For animal species cell tropism, both SARS-CoV and SARS-CoV-2 replicated in non-human primate, cat, rabbit, and pig cells. SARS-CoV, but not SARS-CoV-2, infected and replicated in Rhinolophus sinicus bat kidney cells. SARS-CoV-2 consistently induced significantly delayed and milder levels of cell damage than did SARS-CoV in non-human primate cells (VeroE6, p=0·016; FRhK4, p=0·0004). Interpretation As far as we know, our study presents the first quantitative data for tropism, replication kinetics, and cell damage of SARS-CoV-2. These data provide novel insights into the lower incidence of diarrhoea, decreased disease severity, and reduced mortality in patients with COVID-19, with respect to the pathogenesis and high transmissibility of SARS-CoV-2 compared with SARS-CoV. Funding May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Richard Yu and Carol Yu, Michael Seak-Kan Tong, Respiratory Viral Research Foundation, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund, Chan Yin Chuen Memorial Charitable Foundation, Marina Man-Wai Lee, The Hong Kong Hainan Commercial Association South China Microbiology Research Fund, The Jessie & George Ho Charitable Foundation, Perfect Shape Medical, The Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Diseases and Research Capability on Antimicrobial Resistance for the Department of Health of the Hong Kong Special Administrative Region Government, The Theme-Based Research Scheme of the Research Grants Council, Sanming Project of Medicine in Shenzhen, and The High Level-Hospital Program, Health Commission of Guangdong Province, China.

positive: Coding potential and sequence conservation of SARS-CoV-2 and related animal viruses Abstract In December 2019, a novel human-infecting coronavirus (SARS-CoV-2) was recognized in China. In a few months, SARS-CoV-2 has caused thousands of disease cases and deaths in several countries. Phylogenetic analyses indicated that SARS-CoV-2 clusters with SARS-CoV in the Sarbecovirus subgenus and viruses related to SARS-CoV-2 were identified from bats and pangolins. Coronaviruses have long and complex genomes with high plasticity in terms of gene content. To date, the coding potential of SARS-CoV-2 remains partially unknown. We thus used available sequences of bat and pangolin viruses to determine the selective events that shaped the genome structure of SARS-CoV-2 and to assess its coding potential. By searching for signals of significantly reduced variability at synonymous sites (dS), we identified six genomic regions, one of these corresponding to the programmed −1 ribosomal frameshift. The most prominent signal of dS reduction was observed within the E gene. A genome-wide analysis of conserved RNA structures indicated that this region harbors a putative functional RNA element that is shared with the SARS-CoV lineage. Additional signals of reduced dS indicated the presence of internal ORFs. Whereas the presence ORF9a (internal to N) was previously proposed by homology with a well characterized protein of SARS-CoV, ORF3h (for hypothetical, within ORF3a) was not previously described. The predicted product of ORF3h has 90% identity with the corresponding predicted product of SARS-CoV and displays features suggestive of a viroporin. Finally, analysis of the putative ORF10 revealed high dN/dS (3.82) in SARS-CoV-2 and related coronaviruses. In the SARS-CoV lineage, the ORF is predicted to encode a truncated protein and is neutrally evolving. These data suggest that ORF10 encodes a functional protein in SARS-CoV-2 and that positive selection is driving its evolution. Experimental analyses will be necessary to validate and characterize the coding and non-coding functional elements we identified.

positive: Genetic Diversity and Genomic Epidemiology of SARS-COV-2 in Morocco COVID-A9 is an infection disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), declared as a pandemic due to its rapid expansion worldwide. In this study we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 using 22 virus genome sequences reported by three different laboratories in Morocco till the date 07/06/2020 as well as (40366) virus genomes from all around the world. The SARS-CoV-2 genomes from Moroccan patients revealed 62 mutations of which 30 were missense mutations. The mutations Spike_D614G and NSP12_P323L were present in all the 22 analyzed sequences, followed by N_G204R and N_R203K which occurred in 9 among the 22 sequences. The mutations NSP10_R134S, NSP15_D335N, NSP16_I169L, NSP3_L431H, NSP3_P1292L and Spike_V6F occurred one time in our sequences with no record in other sequence worldwide. These mutations should be investigated to figure out their potential effects on all around the world virulence. Phylogenetic analyses revealed that Moroccan SARS-CoV-2 genomes included 9 viruses pertaining to clade 20A, 9 to clade 20B and 2 to clade 20C. This finding suggest that the epidemic spread in Morocco did not show a predominant SARS-CoV-2 route. For multiple and unrelated introductions of SARS-CoV-2 into Morocco via different routes have occurred, giving rise to the diversity of virus genomes in the country. Furthermore, very likely, the SARS-CoV-2 virus circulated in cryptic way in Morocco starting from the fifteen January before the discovering of the first case the second of March.

positive: COVID-19: An Update on the Epidemiological, Genomic Origin, Phylogenetic study, India centric to Worldwide current status The pandemic spread of novel coronavirus, (SARS-CoV-2) causing CoronaVirus Infectious Diseases (COVID-19) emerged into a global threat for human life causing serious death rates and economic crunch all over the globe. As on April 17, 2020 at 2:00am CEST, there include a total of 2,034,802 confirmed cases for Corona and 1,35,163 deaths worldwide have been reported which includes 212 countries, areas or territories reported by World Health Organization (WHO), in which USA tops 6,32,781 confirmed cases (28,221 deaths) followed by Italy 1,65,155 (21,647 deaths), Spain 1,77,633 (18,579 deaths) and China 84,149 (4,642 deaths). This study aims to compare the genomic nature of SARS-CoV-2 genome reported from Wuhan, China with two Indian isolate genome reported by ICMR-NIV, India. Further Phylogenetic studies performed with coronavirus infecting non-human species like Bats, Duck, and sparrow were compared with Indian and other country whole genome sequences of SARS-CoV2 using MegaX and traced out the association between the human coronavirus with the other species viral genome. In addition, epidemiological reports on COVID-19 among Worldwide and India centric data were compared between April 7, 2020 to April 17, 2020 global data and the number of active cases were increased dramatically in this 10 days period studied, highlighted in the current study.

positive: Genomic variance of the 2019‐nCoV coronavirus There is a rising global concern for the recently emerged novel coronavirus (2019‐nCoV). Full genomic sequences have been released by the worldwide scientific community in the last few weeks to understand the evolutionary origin and molecular characteristics of this virus. Taking advantage of all the genomic information currently available, we constructed a phylogenetic tree including also representatives of other coronaviridae, such as Bat coronavirus (BCoV) and severe acute respiratory syndrome. We confirm high sequence similarity (>99%) between all sequenced 2019‐nCoVs genomes available, with the closest BCoV sequence sharing 96.2% sequence identity, confirming the notion of a zoonotic origin of 2019‐nCoV. Despite the low heterogeneity of the 2019‐nCoV genomes, we could identify at least two hypervariable genomic hotspots, one of which is responsible for a Serine/Leucine variation in the viral ORF8‐encoded protein. Finally, we perform a full proteomic comparison with other coronaviridae, identifying key aminoacidic differences to be considered for antiviral strategies deriving from previous anti‐coronavirus approaches.

positive: Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

positive: An exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (SARS-CoV-2) The city of Wuhan, Hubei province, China, was the origin of a severe pneumonia outbreak in December 2019, attributed to a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]), causing a total of 2761 deaths and 81109 cases (25 February 2020). SARS-CoV-2 belongs to genus Betacoronavirus, subgenus Sarbecovirus. The polyprotein 1ab (pp1ab) remains unstudied thoroughly since it is similar to other sarbecoviruses. In this short communication, we performed phylogenetic-structural sequence analysis of pp1ab protein of SARS-CoV-2. The analysis showed that the viral pp1ab has not changed in most isolates throughout the outbreak time, but interestingly a deletion of 8 aa in the virulence factor nonstructural protein 1 was found in a virus isolated from a Japanese patient that did not display critical symptoms. While comparing pp1ab protein with other betacoronaviruses, we found a 42 amino acid signature that is only present in SARS-CoV-2 (AS-SCoV2). Members from clade 2 of sarbecoviruses have traces of this signature. The AS-SCoV2 located in the acidic-domain of papain-like protein of SARS-CoV-2 and bat-SL-CoV-RatG13 guided us to suggest that the novel 2019 coronavirus probably emerged by genetic drift from bat-SL-CoV-RaTG13. The implication of this amino acid signature in papain-like protein structure arrangement and function is something worth to be explored.

positive: Evolutionary history, potential intermediate animal host, and cross‐species analyses of SARS‐CoV‐2 To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS‐CoV‐2 virus, we reanalyzed virome data sets from pangolins and representative SARS‐related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood‐mapping, and comparative analyses of the genomes. Based on Bayesian time‐scaled phylogenetic analysis using the tip‐dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS‐CoV‐2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10(−3) substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS‐CoV‐2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS‐CoV‐2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS‐CoV‐2 virus, which is responsible for the recent outbreak of COVID‐19, did not come directly from pangolins.

positive: Evolving COVID-19 conundrum and its impact

positive: Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10 - 25 January) 2020. We also applied structural bioinformatics to assess D614G potential impact on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.

positive: Genomic Analysis and Comparative Multiple Sequence of SARS-CoV2. BACKGROUND China announced an outbreak of new coronavirus in the city of Wuhan on December 31, 2019; lash to now, the virus transmission has become pandemic worldwide. Severe cases from Huanan Seafood Wholesale market in Wuhan were confirmed pneumonia with a novel coronavirus. (2019-nCoV). Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies. Thus, we defined the correlation in ten SARS-CoV2 sequences from different countries to analyze the genomic patterns of disease origin and evolution aiming for development new control pandemic processes. METHODS We apply genomic analysis to observe SARS-Co-V2 sequences from GenBank (http:// www.ncbi.nim.nih.gov/genebank/): MN 908947 China, C1), MN985325 (USA:WA, UW), MN996527 (China, C2), MT007544 (Australia:Victoria, A1), MT027064 (USA:CA, UC), MT039890 (South Korea, K1), MT066175 (Taiwan,T1), MT066176 (Taiwan, T2), LC528232 (Japan, J1), and LC528233 (Japan, J2) for genomic sequence alignment an analysis. Multiple Sequence Alignment by Clustalw (https://www.genome.jp/tools-bin/clustalw) web is applied as our alignment tool. RESULTS We analyze ten sequences from NCBI database by genome alignment and find no difference in amino acid sequences within M and N proteins. There are two amino acid variances in Spike protein region. One mutation found from south Korea sequence is verified. Two possible "L" and "S" SNP found in ORF1ab and ORF8 regions are detected CONCLUSION:: We perform genomic analysis and comparative multiple sequence of SARS-CoV-2. Studies about the biological symptoms of SARS-CoV-2 in clinic animal and humans will manipulate an understanding the origin of pandemic crisis.

positive: Ideas for how informaticians can get involved with COVID-19 research The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on population health and wellbeing. Biomedical informatics is central to COVID-19 research efforts and for the delivery of healthcare for COVID-19 patients. Critical to this effort is the participation of informaticians who typically work on other basic science or clinical problems. The goal of this editorial is to highlight some examples of COVID-19 research areas that could benefit from informatics expertise. Each research idea summarizes the COVID-19 application area, followed by an informatics methodology, approach, or technology that could make a contribution. It is our hope that this piece will motivate and make it easy for some informaticians to adopt COVID-19 research projects.

negative: Prevalence and genetic diversity analysis of human coronaviruses among cross-border children BACKGROUND: More than a decade after the outbreak of human coronaviruses (HCoVs) SARS in Guangdong province and Hong Kong SAR of China in 2002, there is still no reoccurrence, but the evolution and recombination of the coronaviruses in this region are still unknown. Therefore, surveillance on the prevalence and the virus variation of HCoVs circulation in this region is conducted. METHODS: A total of 3298 nasopharyngeal swabs samples were collected from cross-border children (<6 years, crossing border between Southern China and Hong Kong SAR) showing symptoms of respiratory tract infection, such as fever (body temperature > 37.5 °C), from 2014 May to 2015 Dec. Viral nucleic acids were analyzed and sequenced to study the prevalence and genetic diversity of the four human coronaviruses. The statistical significance of the data was evaluated with Fisher chi-square test. RESULTS: 78 (2.37%; 95%CI 1.8-2.8%) out of 3298 nasopharyngeal swabs specimens were found to be positive for OC43 (36;1.09%), HKU1 (34; 1.03%), NL63 (6; 0.18%) and 229E (2;0.01%). None of SARS or MERS was detected. The HCoVs predominant circulating season was in transition of winter to spring, especially January and February and NL63 detected only in summer and fall. Complex population with an abundant genetic diversity of coronaviruses was circulating and they shared homology with the published strains (99-100%). Besides, phylogenetic evolutionary analysis indicated that OC43 coronaviruses were clustered into three clades (B,D,E), HKU1 clustered into two clades(A,B) and NL63 clustered into two clades(A,B). Moreover, several novel mutations including nucleotides substitution and the insertion of spike of the glycoprotein on the viral surface were discovered. CONCLUSIONS: The detection rate and epidemic trend of coronaviruses were stable and no obvious fluctuations were found. The detected coronaviruses shared a conserved gene sequences in S and RdRp. However, mutants of the epidemic strains were detected, suggesting continuous monitoring of the human coronaviruses is in need among cross-border children, who are more likely to get infected and transmit the viruses across the border easily, in addition to the general public.

negative: Monophyletic Relationship between Severe Acute Respiratory Syndrome Coronavirus and Group 2 Coronaviruses Although primary genomic analysis has revealed that severe acute respiratory syndrome coronavirus (SARS CoV) is a new type of coronavirus, the different protein trees published in previous reports have provided no conclusive evidence indicating the phylogenetic position of SARS CoV. To clarify the phylogenetic relationship between SARS CoV and other coronaviruses, we compiled a large data set composed of 7 concatenated protein sequences and performed comprehensive analyses, using the maximum-likelihood, Bayesian-inference, and maximum-parsimony methods. All resulting phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between SARS CoV and group 2 CoVs is monophyletic. Relationships among all major groups were well resolved and were supported by all statistical analyses.

negative: CVTree update: a newly designed phylogenetic study platform using composition vectors and whole genomes The CVTree web server (http://tlife.fudan.edu.cn/cvtree) presented here is a new implementation of the whole genome-based, alignment-free composition vector (CV) method for phylogenetic analysis. It is more efficient and user-friendly than the previously published version in the 2004 web server issue of Nucleic Acids Research. The development of whole genome-based alignment-free CV method has provided an independent verification to the traditional phylogenetic analysis based on a single gene or a few genes. This new implementation attempts to meet the challenge of ever increasing amount of genome data and includes in its database more than 850 prokaryotic genomes which will be updated monthly from NCBI, and more than 80 fungal genomes collected manually from several sequencing centers. This new CVTree web server provides a faster and stable research platform. Users can upload their own sequences to find their phylogenetic position among genomes selected from the server's; inbuilt database. All sequence data used in a session may be downloaded as a compressed file. In addition to standard phylogenetic trees, users can also choose to output trees whose monophyletic branches are collapsed to various taxonomic levels. This feature is particularly useful for comparing phylogeny with taxonomy when dealing with thousands of genomes.

negative: Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a “natural knockout” model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10495-020-01614-4) contains supplementary material, which is available to authorized users.

negative: First case of placental infection with SARS-CoV-2 Background: The effects of Covid-19 in pregnancy remain relatively unknown. We present a case of second trimester pregnancy with symptomatic Covid-19 complicated by severe preeclampsia and placental abruption. Methods: We analyzed placenta for the presence of SARS-CoV-2 through molecular and immunohistochemical assays and by and electron microscopy, and we measured the maternal antibody response in blood to this infection. Results: SARS-CoV-2 localized predominantly to syncytiotrophoblast cells at the maternal-fetal interface of the placenta. Histological examination of the placenta revealed a dense macrophage infiltrate, but no evidence for vasculopathy typically associated with preeclampsia. Conclusion: This case demonstrates, for the first time, SARS-CoV-2 invasion of the placenta, highlighting the potential for severe morbidity among pregnant women with Covid-19.

negative: Genomic epidemiology, evolution, and transmission dynamics of porcine deltacoronavirus The emergence of severe acute respiratory syndrome virus (SARS)-CoV-2 has shown once again that coronaviruses (CoVs) in animals are potential sources for epidemics in humans. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen of swine with a worldwide distribution. Here, we implemented and described an approach to analyze the epidemiology of PDCoV following its emergence in the pig population. We performed an integrated analysis of full genome sequence data from 21 newly-sequenced viruses, along with comprehensive epidemiological surveillance data collected globally over the last 15 years. We found four distinct phylogenetic lineages of PDCoV, which differ in their geographic circulation patterns. Interestingly, we identified more frequent intra- and inter-lineage recombination and higher virus genetic diversity in the Chinese lineages compared with the US lineage where pigs are raised in different farming systems and ecological environments. Most recombination breakpoints are located in the ORF1ab gene rather than in genes encoding structural proteins. We also identified five amino acids under positive selection in the spike protein suggesting a role for adaptive evolution. According to structural mapping, three positively selected sites are located in the N-terminal domain of the S1 subunit, which is the most likely involved in binding to a carbohydrate receptor, while the other two are located in or near the fusion peptide of the S2 subunit and thus might affect membrane fusion. Finally, our phylogeographic investigations highlighted notable South-North transmission as well as frequent long-distance dispersal events in China that could implicate human-mediated transmission. Our findings provide new insights into the evolution and dispersal of PDCoV that contribute to our understanding of the critical factors involved in coronaviruses emergence. SIGNIFICANCE: Coronaviruses (CoVs) exhibit a pronounced propensity for interspecies transmission as illustrated by important emerging viruses in humans such as severe acute respiratory syndrome virus (SARS)-CoV and Middle East respiratory syndrome virus (MERS)-CoV, as well as the recent SARS-CoV-2 that is causing a major human pandemic. A better understanding of the evolutionary process leading to viral emergence may help unravel the principles of cross-species transmission and therefore assist in preventing future introductions into new host populations. Here, we examined the evolution and spread of an emerging coronavirus that likely transmitted from birds to pigs and caused sustained epidemics in Asia and the United States. We integrated viral full genome sequence analysis and comprehensive surveillance data to examine the evolution of emerging porcine deltacoronavirus (PDCoV), and in particular the factors driving ongoing circulation and recent cases in China. We found evidence for adaptive evolution of this emerging virus in the absence of vaccine immune pressure, with important amino acids under positive selection, as well as frequent inter- and intra-lineage recombination events in the Asian lineages. Breeding patterns and ecological environment could play an important role in swine coronavirus evolution, leading to differences in recombination and population diversity. Using phylogeographic approaches, we further shed light on the dispersal history and dynamics of the China lineage of PDCoV. Our study provides a deeper understanding of the geographic dispersal of genetic lineages of PDCoV in China and suggests that southern China acted as a source of transmission. Modeling how the virus spread in China will allow the prioritization of areas for swine coronavirus surveillance in the future. These findings have important implications for preventing the emergence of coronaviruses in livestock and for controlling their spread in Asian countries.

negative: Comment on “Genetic variants and source of introduction of SARS‐CoV‐2 in South America” I read with great interest a recent study by Poterico and Mestanza1 who described mutations in 30 SARS-CoV-2 genomes from South American countries (Argentina, Brazil, Chile, Colombia, Ecuador, and Peru) This article is protected by copyright. All rights reserved.

negative: Challenges and management options of tubo-ovarian cancer during the SARS-CoV-2 pandemic

negative: Immunoinformatic analysis of the SARS-CoV-2 Envelope protein as a strategy to assess cross-protection against COVID-19 Envelope protein of coronaviruses is a structural protein existing in both monomeric and homo-pentameric form. It has been related to a multitude of roles including virus infection, replication, dissemination and immune response stimulation. In the present study, we employed an immunoinformatic approach to investigate the major immunogenic domains of the SARS-CoV-2 envelope protein and map them among the homologue proteins of coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Also, when not available, we predicted the envelope protein structural folding and mapped SARS-CoV-2 epitopes. Envelope sequences alignment provides evidence of high sequence homology for some of the investigated virus specimens; while the structural mapping of epitopes resulted in the interesting maintenance of the structural folding and epitope sequence localization also in the envelope proteins scoring a lower alignment score. In line with the One-Health approach, our evidences provide a molecular structural rationale for a potential role of taxonomically related coronaviruses in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.

negative: Positional Correlation Natural Vector: A Novel Method for Genome Comparison Advances in sequencing technology have made large amounts of biological data available. Evolutionary analysis of data such as DNA sequences is highly important in biological studies. As alignment methods are ineffective for analyzing large-scale data due to their inherently high costs, alignment-free methods have recently attracted attention in the field of bioinformatics. In this paper, we introduce a new positional correlation natural vector (PCNV) method that involves converting a DNA sequence into an 18-dimensional numerical feature vector. Using frequency and position correlation to represent the nucleotide distribution, it is possible to obtain a PCNV for a DNA sequence. This new numerical vector design uses six suitable features to characterize the correlation among nucleotide positions in sequences. PCNV is also very easy to compute and can be used for rapid genome comparison. To test our novel method, we performed phylogenetic analysis with several viral and bacterial genome datasets with PCNV. For comparison, an alignment-based method, Bayesian inference, and two alignment-free methods, feature frequency profile and natural vector, were performed using the same datasets. We found that the PCNV technique is fast and accurate when used for phylogenetic analysis and classification of viruses and bacteria.

negative: Prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in South Korea, 2014–2016 Porcine deltacoronavirus (PDCoV) is a newly emerged enterotropic swine coronavirus that causes enteritis and diarrhoea in piglets. Here, a nested reverse transcription (RT)‐PCR approach for the detection of PDCoV was developed to identify and characterize aetiologic agent(s) associated with diarrhoeal diseases in piglets in South Korea. A PCR‐based method was applied to investigate the presence of PDCoV in 683 diarrhoeic samples collected from 449 commercial pig farms in South Korea from January 2014 to December 2016. The molecular‐based survey indicated a relatively high prevalence of PDCoV (19.03%) in South Korea. Among those, the monoinfection of PDCoV (9.66%) and co‐infection of PDCoV (6.30%) with porcine epidemic diarrhoea (PEDV) were predominant in diarrhoeal samples. The full‐length genomes or the complete spike genes of the most recent strains identified in 2016 (KNU16‐07, KNU16‐08 and KNU16‐11) were sequenced and analysed to characterize PDCoV currently prevalent in South Korea. We found a single insertion‐deletion signature and dozens of genetic changes in the spike (S) genes of the KNU16 isolates. Phylogenetic analysis based on the entire genome and spike protein sequences of these strains indicated that they are most closely related to other Korean isolates grouped with the US strains. However, Korean PDCoV strains formed different branches within the same cluster, implying continuous evolution in the field. Our data will advance the understanding of the molecular epidemiology and evolutionary characteristics of PDCoV circulating in South Korea.

negative: Evidence of significant natural selection in the evolution of SARS-CoV-2 in bats, not humans RNA viruses are proficient at switching to novel host species due to their fast mutation rates. Implicit in this assumption is the need to evolve adaptations in the new host species to exploit their cells efficiently. However, SARS-CoV-2 has required no significant adaptation to humans since the pandemic began, with no observed selective sweeps to date. Here we contrast the role of positive selection and recombination in the Sarbecoviruses in horseshoe bats to SARS-CoV-2 evolution in humans. While methods can detect some evidence for positive selection in SARS-CoV-2, we demonstrate these are mostly due to recombination and sequencing artefacts. Purifying selection is also substantially weaker in SARS-CoV-2 than in the related bat Sarbecoviruses. In comparison, our results show evidence for positive, specifically episodic selection, acting on the bat virus lineage SARS-CoV-2 emerged from. This signature of selection can also be observed among synonymous substitutions, for example, linked to ancestral CpG depletion on this bat lineage. We show the bat virus RmYN02 has recombinant CpG content in Spike pointing to coinfection and evolution in bats without involvement of other species. Our results suggest the non-human progenitor of SARS-CoV-2 was capable of human-human transmission as a consequence of its natural evolution in bats.

negative: Time-series analyses of directional sequence changes in SARS-CoV-2 genomes and an efficient search method for advantageous mutations for growth in human cells We first conducted time-series analysis of mono- and dinucleotide composition for over 10,000 SARS-CoV-2 genomes, as well as over 1500 Zaire ebolavirus genomes, and found clear time-series changes in the compositions on a monthly basis, which should reflect viral adaptations for efficient growth in human cells. We next developed a sequence alignment free method that extensively searches for advantageous mutations and rank them in an increase level for their intrapopulation frequency. Time-series analysis of occurrences of oligonucleotides of diverse lengths for SARS-CoV-2 genomes revealed seven distinctive mutations that rapidly expanded their intrapopulation frequency and are thought to be candidates of advantageous mutations for the efficient growth in human cells.

negative: The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin

negative: Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003 BACKGROUND: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. METHODS: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. RESULTS: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14(th )October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10(-6 )nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. CONCLUSIONS: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

negative: Epidemiology and Phylogenetic Analysis of Viral Respiratory Infections in Vietnam Acute respiratory infections (ARIs) impose a major public health burden on fragile healthcare systems of developing Southeast Asian countries such as Vietnam. The epidemiology, genetic diversity and transmission patterns of respiratory viral pathogens that circulate in this region are not well characterized. We used RT-PCR to screen for 14 common respiratory viruses in nasal/throat samples from 4326 ARI patients from 5 sites in Vietnam during 2012–2016. 64% of patients tested positive for viruses; 14% tested positive multiple co-infecting viruses. The most frequently detected viruses were Respiratory syncytial virus (RSV, 23%), Human Rhinovirus (HRV, 13%), Influenza A virus (IAV, 11%) and Human Bocavirus (HBoV, 7%). RSV infections peaked in July to October, were relatively more common in children <1 year and in the northernmost hospital. IAV infections peaked in December to February and were relatively more common in patients >5 years in the central region. Coinfection with IAV or RSV was associated with increased disease severity compared with patients only infected with HBoV or HRV. Over a hundred genomes belonging to 13 families and 24 genera were obtained via metagenomic sequencing, including novel viruses and viruses less commonly associated with ARIs. Phylogenetic and phylogeographic analyses further indicated that neighboring countries were the most likely source of many virus lineages causing ARIs in Vietnam and estimated the period that specific lineages have been circulating. Our study illustrates the value of applying the state-of-the-art virus diagnostic methods (multiplex RT-PCR and metagenomic sequencing) and phylodynamic analyses at a national level to generate an integrated picture of viral ARI epidemiology.

negative: Cyclosporine therapy in cytokine storm due to coronavirus disease 2019 (COVID-19)

negative: Detection of Alphacoronavirus in velvety free-tailed bats (Molossus molossus) and Brazilian free-tailed bats (Tadarida brasiliensis) from urban area of Southern Brazil A survey was carried out in search for bat coronaviruses in an urban maternity roost of about 500 specimens of two species of insectivorous bats, Molossus molossus and Tadarida brasiliensis, in Southern Brazil. Twenty-nine out of 150 pooled fecal samples tested positive by reverse transcription-PCR contained fragments of the RNA-dependent RNA polymerase gene of coronavirus-related viruses. The sequences clustered along with bat alphacoronaviruses, forming a subcluster within this group. Our findings point to the need for risk assessment and continued surveillance of coronavirus infections of bats in Brazil. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-013-0899-x) contains supplementary material, which is available to authorized users.

negative: Association of national Bacille Calmette-Guerin vaccination policy with COVID-19 epidemiology: an ecological study in 78 countries A possible association between national Bacille Calmette-Guerin (BCG) vaccination policy and lower COVID-19 incidence has been suggested in some preprint papers. Using publicly accessible databases, I explored associations of national BCG vaccination policy with COVID-19 epidemiology in 78 countries. Data collection was conducted from April 25 to May 5, 2020. I compared countries that have a current universal BCG vaccination policy (BCG countries), with countries that currently lack such a policy (non-BCG countries). The mixed effect model revealed national BCG policy decreases in the country-specific risk of death by COVID-19, correspond to odds ratio of 0.446 (95% confidence intervals 0.323 - 0.614, P =1x10-5). In BCG countries, the case increase rate was attenuated marginally by 25.4% (95% CI 3.0 to 42.7, P=0.029) as compared with those of the non-BCG countries. Although the protective mechanism of BCG vaccination against COVID-19 remains unknown, further laboratory and clinical research should be warranted.

negative: SARS associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting Abstract The sudden appearance and potential lethality of severe acute respiratory syndrome associated coronavirus (SARS-CoV) in humans has focused attention on understanding its origins. Here, we assess phylogenetic relationships for the SARS-CoV lineage as well as the history of host-species shifts for SARS-CoV and other coronaviruses. We used a Bayesian phylogenetic inference approach with sliding window analyses of three SARS-CoV proteins: RNA dependent RNA polymerase (RDRP), nucleocapsid (N) and spike (S). Conservation of RDRP allowed us to use a set of Arteriviridae taxa to root the Coronaviridae phylogeny. We found strong evidence for a recombination breakpoint within SARS-CoV RDRP, based on different, well supported trees for a 5′ fragment (supporting SARS-CoV as sister to a clade including all other coronaviruses) and a 3′ fragment (supporting SARS-CoV as sister to group three avian coronaviruses). These different topologies are statistically significant: the optimal 5′ tree could be rejected for the 3′ region, and the optimal 3′ tree could be rejected for the 5′ region. We did not find statistical evidence for recombination in analyses of N and S, as there is little signal to differentiate among alternative trees. Comparison of phylogenetic trees for 11 known host-species and 36 coronaviruses, representing coronavirus groups 1–3 and SARS-CoV, based on N showed statistical incongruence indicating multiple host-species shifts for coronaviruses. Inference of host-species associations is highly sensitive to sampling and must be considered cautiously. However, current sampling suggests host-species shifts between mouse and rat, chicken and turkey, mammals and manx shearwater, and humans and other mammals. The sister relationship between avian coronaviruses and the 3′ RDRP fragment of SARS-CoV suggests an additional host-species shift. Demonstration of recombination in the SARS-CoV lineage indicates its potential for rapid unpredictable change, a potentially important challenge for public health management and for drug and vaccine development.

negative: First detection and genomic characteristics of bovine torovirus in dairy calves in China Bovine torovirus (BToV) is a diarrhea-causing pathogen. In this study, 92 diarrheic fecal samples from five farms in four provinces in China were collected and tested for BToV using a RT-PCR assay, and 21.73% samples were found to be BToV positive. Moreover, two complete BToV genome sequences (MN073058 and MN073059) were obtained from the clinical samples, which were 28,297 and 28,301 nucleotides in length, respectively. Sequence analysis showed that the two isolates shared 10 identical amino acid mutations in the S protein compared to the complete S sequences of BToV available in the GenBank database. In addition, seven consecutive amino acid mutations were found from aa 1,486 to 1,492 in the S protein of isolate MN073058. Moreover, the two isolates shared one identical amino acid mutation in the receptor binding sites of the HE protein. To the best of our knowledge, this is the first report on the epidemic and genomic characterization of BToV in China, which is helpful for further understanding the genetic evolution of BToV. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00705-020-04657-9) contains supplementary material, which is available to authorized users.

negative: Human coronaviruses: Clinical features and phylogenetic analysis Abstract Strains of human coronavirus (HCoV), namely HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1, primarily infect the upper respiratory and gastrointestinal tracts and are the most common cause of non-rhinovirus-induced common cold in humans. Although the manifestations of coronavirus infection (i.e., rhinorrhea, sneezing, cough, nasal obstruction, and bronchitis) are generally self-limiting in healthy adults, certain strains such as HCoV-NL63 and HCoV-HKU1 can cause severe lower respiratory tract infection and febrile seizure, especially in infants, people of advanced age, and immunocompromised hosts. In 2003, a novel HCoV strain was identified as the causative agent of the severe acute respiratory syndrome (SARS) epidemic that began in Asia in 2002. The strain has hence been referred to as SARS-CoV. In addition, as recently as September 2012, another novel HCoV, human betacoronavirus 2c EMC2012, was identified as being the cause of fever, renal failure, pneumonia, and severe respiratory distress in two patients in the Middle East. Phylogenetic analysis has revealed highly conserved sequences of ORF1ab, spike, nucleocapsid, and envelope protein genes, but not membrane protein genes, between human betacoronavirus 2c EMC2012 and SARS-CoV. This review focuses on the differences in the genomes of certain HCoV strains, the pathogenesis of said strains, and recent developments in the establishment of therapeutic agents that might aid in the treatment of patients with such infections.

negative: Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine [Image: see text] Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

negative: First Identification of Human Adenovirus Subtype 21a in China With MinION and Illumina Sequencers Human adenoviruses (HAdVs) have been demonstrated to cause a diversity of diseases among children and adults. The circulation of human adenovirus type 21 (HAdV21) has been mainly documented within closed environments in several countries. Nonetheless, respiratory infections or outbreaks due to HAdV21 have never been reported in China. MinION and Illumina platforms were employed to identify the potential pathogen from a throat swab. Discrepancies between MinION and Illumina sequencing were validated and corrected via polymerase chain reaction (PCR). Genomic characterization and recombinant event detection were then performed. Among the 35,466 high-quality MinION reads, a total of 5,999 reads (16.91%) could be aligned to HAdV21 reference genomes (genome sizes ≈35.3 kb), among which 20 had a length of >30 kb. A genome sequence assembled from MinION reads was further classified as HAdV subtype 21a. Random downsampling revealed as few as 500 nanopore reads could cover ≥96.49% of current genome. Illumina sequencing displayed good consistency (pairwise nucleotide identity = 99.91%) with MinION sequencing but with 31 discrepancies that were further validated and confirmed by PCR coupled with Sanger sequencing. Restriction enzymes such as BamHI and KpnI were able to distinguish the present genome from HAdV21 prototype and HAdV21b. Phylogenetic analysis employing whole-genome sequences placed our genome with members only from subtype 21a. Common features among HAdV21a strains were identified, including polymorphisms discovered in penton and 100 kDa hexon assembly–associated proteins and a recombinant event in the E4 gene. Using MinION and Illumina sequencers, we identified the first HAdV21a strain from China, which could provide key genomic data for disease control and epidemiological investigations.

negative: Management of CLL patients early in the COVID‐19 pandemic: An international survey of CLL experts

negative: Phylogenetic analysis and sequence comparisons of structural and non-structural SARS coronavirus proteins in Taiwan Abstract Taiwan experienced a large number of severe acute respiratory syndrome (SARS) viral infections between March and July 2003; by September of that year, 346 SARS cases were confirmed by RT-PCR or serological tests. In order to better understand evolutionary relationships among SARS coronaviruses (SCoVs) from different international regions, we performed phylogenetic comparisons of full-length genomic and protein sequences from 45 human SCoVs (including 12 from Taiwan) and two civet SCoVs. All the Taiwanese SARS-CoV strains which associated with nosocomial infection formed a monophyletic clade within the late phase of the SARS epidemic. This Taiwanese clade could be further divided into two epidemic waves. Taiwan SCoVs in the first wave clustered with three isolates from the Amoy Gardens housing complex in Hong Kong indicating their possible origin. Of the 45 human SCoVs, one isolate from Guangdong province, China, exhibited an extra 29-nucleotide fragment between Orf 10 and Orf 11—similar to the civet SCoV genome. Nucleotide and protein sequence comparisons suggested that all SCoVs of late epidemic came from human-to-human transmission, while certain SCoVs of early epidemic might have originated in animals.

negative: Detection and characterization of diverse alpha- and betacoronaviruses from bats in China Bats have been implicated as important reservoir hosts of alpha- and betacoronaviruses. In this study, diverse coronaviruses (CoVs) were detected in 50 of 951 (positive rate 5.3%) intestinal specimens of eight bat species collected in four provinces and the Tibet Autonomous Region of China by pan-coronavirus RT-PCR screening. Based on 400-nt RNA-dependent RNA polymerase (RdRP) sequence analysis, eight belonged to genus Alphacoronavirus and 42 to Betacoronavirus. Among the 50 positive specimens, thirteen gave rise to CoV full-length RdRP gene amplification for further sequence comparison, of which three divergent sequences (two from a unreported province) were subjected to full genome sequencing. Two complete genomes of betacoronaviruses (JTMC15 and JPDB144) and one nearly-complete genome of alphacoronavirus (JTAC2) were sequenced and their genomic organization predicted. The present study has identified additional numbers of genetically diverse bat-borne coronaviruses with a wide distribution in China. Two new species of bat CoV, identified through sequence comparison and phylogenetic analysis, are proposed. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12250-016-3727-3 and is accessible for authorized users.

negative: Genome analysis with the conditional multinomial distribution profile Abstract The focus of the research is on the analysis of genome sequences. Based on the inter-nucleotide distance sequence, we propose the conditional multinomial distribution profile for the complete genomic sequence. These profiles can be used to define a very simple, computationally efficient, alignment-free, distance measure that reflects the evolutionary relationships between genomic sequences. We use this distance measure to classify chromosomes according to species of origin, to build the phylogenetic tree of 24 complete genome sequences of coronaviruses. Our results demonstrate the new method is powerful and efficient.

negative: Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach The sudden emergence of severe respiratory disease, caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently become a public health emergency. Genome sequence analysis of SARS-CoV-2 revealed its close resemblance to the earlier reported SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). However, initial testing of the drugs used against SARS-CoV and MERS-CoV has been ineffective in controlling SARS-CoV-2. The present study highlights the genomic, proteomic, pathogenesis, and therapeutic strategies in SARS-CoV-2 infection. We have carried out sequence analysis of potential drug target proteins in SARS-CoV-2 and, compared them with SARS-CoV and MERS viruses. Analysis of mutations in the coding and non-coding regions, genetic diversity, and pathogenicity of SARS-CoV-2 has also been done. A detailed structural analysis of drug target proteins has been performed to gain insights into the mechanism of pathogenesis, structure-function relationships, and the development of structure-guided therapeutic approaches. The cytokine profiling and inflammatory signalling are different in the case of SARS-CoV-2 infection. We also highlighted possible therapies and their mechanism of action followed by clinical manifestation. Our analysis suggests a minimal variation in the genome sequence of SARS-CoV-2, may be responsible for a drastic change in the structures of target proteins, which makes available drugs ineffective.

negative: Genome sequencing and phylogenetic analysis of Banna virus (genus Seadornavirus, family Reoviridae) isolated from Culicoides In an investigation of blood-sucking insects and arboviruses, a virus (YN12243) was isolated from Culicoides samples collected in the Sino-Burmese border region of Yunnan Province, China. The virus caused cytopathic effect (CPE) in C6/36 cells and passaged stably. Polyacrylamide gel analysis showed that the genome of YN12243 was composed of 12 segments of double-stranded RNA (dsRNA), with a distribution pattern of 6-6. The nucleotide and amino acid sequences of the coding region (1‒12 segments) were 17,803 bp and 5,925 amino acids in length, respectively. The phylogenetic analysis of VP1 protein (RdRp) revealed that YN12243 belonged to genus Seadornavirus of family Reoviridae, and further analysis indicated that YN12243 belongs to the Banna virus (BAV) genotype A2. Additionally, YN12243 was located in the same evolutionary cluster as BAV strains isolated from different mosquito species, suggesting that the BAV isolated from Culicoides does not have species barriers. These results indicate that Culicoides can also be a vector for BAV. In view of the hematophagous habits of Culicoides on cattle, horses, deer, and other large animals, as well as the possibility of spreading and causing a variety of animal arboviral diseases, it is important to improve infection detection and monitor the BAV in large livestock.

negative: Evidence of Increasing Diversification of Emerging SARS-CoV-2 Strains BACKGROUND: On January 30th, 2020, an outbreak of atypical pneumonia caused by a novel Betacoronavirus (ßCoV), named SARS-CoV-2, was declared a public health emergency of international concern by the World Health Organization. For this reason, a detailed evolutionary analysis of SARS-CoV-2 strains currently circulating in different geographic regions of the world was performed. METHODS: A compositional analysis as well as a Bayesian coalescent analysis of complete genome sequences of SARS-CoV-2 strains recently isolated in Europe, North America, South America and Asia was performed. RESULTS: The results of these studies revealed a diversification of SARS-CoV-2 strains in three different genetic clades. Co-circulation of different clades in different countries, as well as different genetic lineages within different clades were observed. The time of the most recent common ancestor (tMRCA) was established to be around November 1, 2019. A mean rate of evolution of 6.57 x 10-4 substitutions per site per year was found. A significant migration rate per genetic lineage per year from Europe to South America was also observed. CONCLUSION: The results of these studies revealed an increasing diversification of SARS-CoV-2 strains. High evolutionary rates and fast population growth characterizes the population dynamics of SARS-CoV-2 strains. This article is protected by copyright. All rights reserved.

negative: A chilblain epidemic during the COVID-19 pandemic. A sign of natural resistance to SARS-CoV-2?

negative: Comparing the Binding Interactions in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV [Image: see text] SARS-CoV-2, since emerging in Wuhan, China, has been a major concern because of its high infection rate and has left more than six million infected people around the world. Many studies endeavored to reveal the structure of the SARS-CoV-2 compared to the SARS-CoV, in order to find solutions to suppress this high infection rate. Some of these studies showed that the mutations in the SARS-CoV spike (S) protein might be responsible for its higher affinity to the ACE2 human cell receptor. In this work, we used molecular dynamics simulations and Monte Carlo sampling to compare the binding affinities of the S proteins of SARS-CoV and SARS-CoV-2 to the ACE2. Our results show that the protein surface of the ACE2 at the receptor binding domain (RBD) exhibits negative electrostatic potential, while a positive potential is observed for the S proteins of SARS-CoV/SARS-CoV-2. In addition, the binding energies at the interface are slightly higher for SARS-CoV-2 because of enhanced electrostatic interactions. The major contributions to the electrostatic binding energies result from the salt bridges forming between R426 and ACE-2-E329 in the case of SARS-CoV and K417 and ACE2-D30 in the SARS-CoV-2. In addition, our results indicate that the enhancement in the binding energy is not due to a single mutant but rather because of the sophisticated structural changes induced by all these mutations together. This finding suggests that it is implausible for the SARS-CoV-2 to be a lab-engineered virus.

negative: Overview of therapeutic drug research for COVID-19 in China Since the outbreak of novel coronavirus pneumonia (COVID-19) in December 2019, more than 2,500,000 people worldwide have been diagnosed with SARS-CoV-2 as of April 22. In response to this epidemic, China has issued seven trial versions of diagnosis and treatment protocol for COVID-19. According to the information that we have collected so far, this article provides an overview of potential therapeutic drugs and compounds with much attention, including favipiravir and hydroxychloroquine, as well as traditional Chinese medicine, which have been reported with good clinical treatment effects. Moreover, with further understanding of SARS-CoV-2 virus, new drugs targeting specific SARS-CoV-2 viral components arise and investigations on these novel anti-SARS-CoV-2 agents are also reviewed.

negative: Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60–70%. Improved sensitivity is, therefore, urgently required. METHODS: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances. RESULTS: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56% for NIID-N1, 61% for YCH-N1, and 94% for NIID-N2, and 100% negative percent agreements for NIID-N1, YCH-N1 and NIID-N2. CONCLUSION: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.

negative: [Clinical features of children with SARS-CoV-2 infection: an analysis of 115 cases] OBJECTIVE: To study the clinical features of children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: A retrospective analysis was performed for the clinical data of 115 children who were diagnosed with SARS-CoV-2 infection in the Wuhan Children's Hospital, including general information, history of close contact with individuals of SARS-CoV-2 infection, early clinical symptoms, laboratory examination results, and lung CT results. RESULTS: Among the 115 children, there were 73 boys (63.5%) and 42 girls (36.5%), with a male/female ratio of 1:0.58. Of the 115 children, 105 (91.3%) had a definite history of close contact with individuals of SARS-CoV-2-infection. An increase in alanine aminotransferase was observed in 11 children (9.6%) and an increase in CK-MB was found in 34 children (29.6%). As for clinical symptoms, 29 children (25.2%) had fever, 47 (40.9%) had respiratory symptoms (including cough, rhinorrhea, and nasal congestion), and 61 (53.0%) were asymptomatic. Lung CT findings showed ground glass opacity, fiber opacities, patchy changes, and pulmonary consolidation in 49 children (42.6%), among whom 2 children had "white lung"; 39 children (33.9%) only had lung texture enhancement and 27 children (23.5%) had no pulmonary imaging changes. Among the 115 children, 3 were critically ill, among whom 1 had been cured and the other 2 were under continuous treatment. CONCLUSIONS: Most of the children with SARS-CoV-2 infection have a close contact history. Critical cases are rare and there is a high proportion of asymptomatic infection.

negative: Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.

negative: Machine intelligence design of 2019-nCoV drugs Wuhan coronavirus, called 2019-nCoV, is a newly emerged virus that infected more than 9692 people and leads to more than 213 fatalities by January 30, 2020. Currently, there is no effective treatment for this epidemic. However, the viral protease of a coronavirus is well-known to be essential for its replication and thus is an effective drug target. Fortunately, the sequence identity of the 2019-nCoV protease and that of severe-acute respiratory syndrome virus (SARS-CoV) is as high as 96.1%. We show that the protease inhibitor binding sites of 2019-nCoV and SARS-CoV are almost identical, which means all potential anti-SARS-CoV chemotherapies are also potential 2019-nCoV drugs. Here, we report a family of potential 2019-nCoV drugs generated by a machine intelligence-based generative network complex (GNC). The potential effectiveness of treating 2019-nCoV by using some existing HIV drugs is also analyzed.

negative: What to expect from antibody assays of SARS-CoV-2?

negative: Molecular Evolution of MERS Coronavirus: Dromedaries as a Recent Intermediate Host or Long-Time Animal Reservoir? While dromedary camels are the immediate animal source of MERS coronavirus (MERS-CoV) infection, the evolutionary origin of MERS-CoV remains obscure. We analyzed 219 camel and human MERS-CoV genome sequences available in GenBank. Phylogenetic analysis showed that 5 and 214 strains belong to clade A and B, respectively, with clade A further divided into lineage A1 (3 human strains) and lineage A2 (2 camel strains), and clade B divided into B1 to B6 (each containing both human and camel strains). Recombination analysis showed potential recombination events in five strains from dromedaries in Saudi Arabia, with recombination between lineage B5 and B3 in four strains, and between lineage B3 and B4 in one strain. The spike protein showed the highest number of amino acid substitutions, especially between A2 and other lineages, and contained positively selected codons. Notably, codon 1020 was positively selected among B and B5 strains, and can distinguish between clade A (Q1020) and B (R1020/H1020) strains, suggesting that this residue may play a role in the evolution of S protein during divergence of different lineages. The time of the most recent common ancestor of all MERS-CoV was dated to approximately 2010. The implications on the role of camels in the evolution of MERS-CoV are discussed.

negative: Comparative analysis estimates the relative frequencies of co-divergence and cross-species transmission within viral families The cross-species transmission of viruses from one host species to another is responsible for the majority of emerging infections. However, it is unclear whether some virus families have a greater propensity to jump host species than others. If related viruses have an evolutionary history of co-divergence with their hosts there should be evidence of topological similarities between the virus and host phylogenetic trees, whereas host jumping generates incongruent tree topologies. By analyzing co-phylogenetic processes in 19 virus families and their eukaryotic hosts we provide a quantitative and comparative estimate of the relative frequency of virus-host co-divergence versus cross-species transmission among virus families. Notably, our analysis reveals that cross-species transmission is a near universal feature of the viruses analyzed here, with virus-host co-divergence occurring less frequently and always on a subset of viruses. Despite the overall high topological incongruence among virus and host phylogenies, the Hepadnaviridae, Polyomaviridae, Poxviridae, Papillomaviridae and Adenoviridae, all of which possess double-stranded DNA genomes, exhibited more frequent co-divergence than the other virus families studied here. At the other extreme, the virus and host trees for all the RNA viruses studied here, particularly the Rhabdoviridae and the Picornaviridae, displayed high levels of topological incongruence, indicative of frequent host switching. Overall, we show that cross-species transmission plays a major role in virus evolution, with all the virus families studied here having the potential to jump host species, and that increased sampling will likely reveal more instances of host jumping.

negative: Relationship of SARS-CoV to other pathogenic RNA viruses explored by tetranucleotide usage profiling BACKGROUND: The exact origin of the cause of the Severe Acute Respiratory Syndrome (SARS) is still an open question. The genomic sequence relationship of SARS-CoV with 30 different single-stranded RNA (ssRNA) viruses of various families was studied using two non-standard approaches. Both approaches began with the vectorial profiling of the tetra-nucleotide usage pattern V for each virus. In approach one, a distance measure of a vector V, based on correlation coefficient was devised to construct a relationship tree by the neighbor-joining algorithm. In approach two, a multivariate factor analysis was performed to derive the embedded tetra-nucleotide usage patterns. These patterns were subsequently used to classify the selected viruses. RESULTS: Both approaches yielded relationship outcomes that are consistent with the known virus classification. They also indicated that the genome of RNA viruses from the same family conform to a specific pattern of word usage. Based on the correlation of the overall tetra-nucleotide usage patterns, the Transmissible Gastroenteritis Virus (TGV) and the Feline CoronaVirus (FCoV) are closest to SARS-CoV. Surprisingly also, the RNA viruses that do not go through a DNA stage displayed a remarkable discrimination against the CpG and UpA di-nucleotide (z = -77.31, -52.48 respectively) and selection for UpG and CpA (z = 65.79,49.99 respectively). Potential factors influencing these biases are discussed. CONCLUSION: The study of genomic word usage is a powerful method to classify RNA viruses. The congruence of the relationship outcomes with the known classification indicates that there exist phylogenetic signals in the tetra-nucleotide usage patterns, that is most prominent in the replicase open reading frames.

negative: Genomic and evolutionary inferences between American and global strains of porcine epidemic diarrhea virus Abstract Porcine epidemic diarrhea virus (PEDV) has caused severe economic losses both recently in the United States (US) and historically throughout Europe and Asia. Traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between PEDV strains. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n =126) to determine the most variable genomic areas. Our results indicate high levels of variation within the ORF1 and spike regions while the C-terminal domains of structural genes were highly conserved. Analysis of the Receptor Binding Domains in the spike gene revealed a limited number of amino acid substitutions in US strains compared to Asian strains. Phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. These finding suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV.

negative: Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility BACKGROUND: A physiological small animal model that resembles COVID-19 with low mortality is lacking. METHODS: Molecular docking on the binding between angiotensin-converting enzyme 2 (ACE2) of common laboratory mammals and the receptor-binding domain of the surface spike protein of SARS-CoV-2 suggested that the golden Syrian hamster is an option. Virus challenge, contact transmission, and passive immunoprophylaxis were performed. Serial organ tissues and blood were harvested for histopathology, viral load and titre, chemokine/cytokine assay, and neutralising antibody titre. RESULTS: The Syrian hamster could be consistently infected by SARS-CoV-2. Maximal clinical signs of rapid breathing, weight loss, histopathological changes from the initial exudative phase of diffuse alveolar damage with extensive apoptosis to the later proliferative phase of tissue repair, airway and intestinal involvement with virus nucleocapsid protein expression, high lung viral load, and spleen and lymphoid atrophy associated with marked cytokine activation were observed within the first week of virus challenge. The lung virus titre was between 10(5)-10(7) TCID(50)/g. Challenged index hamsters consistently infected naïve contact hamsters housed within the same cage, resulting in similar pathology but not weight loss. All infected hamsters recovered and developed mean serum neutralising antibody titre ≥1:427 fourteen days post-challenge. Immunoprophylaxis with early convalescent serum achieved significant decrease in lung viral load but not in lung pathology. No consistent non-synonymous adaptive mutation of the spike was found in viruses isolated from infected hamsters. CONCLUSIONS: Besides satisfying the Koch’s postulates, this readily available hamster model is an important tool for studying transmission, pathogenesis, treatment, and vaccination against SARS-CoV-2.

negative: Chaos game representation dataset of SARS-CoV-2 genome Abstract As of April 16, 2020, the novel coronavirus disease (called COVID-19) spread to more than 185 countries/regions with more than 142,000 deaths and more than 2,000,000 confirmed cases. In the bioinformatics area, one of the crucial points is the analysis of the virus nucleotide sequences using approaches such as data stream, digital signal processing, and machine learning techniques and algorithms. However, to make feasible this approach, it is necessary to transform the nucleotide sequences string to numerical values representation. Thus, the dataset provides a chaos game representation (CGR) of SARS-CoV-2 virus nucleotide sequences. The dataset provides the CGR of 100 instances of SARS-CoV-2 virus, 11540 instances of other viruses from the Virus-Host DB dataset, and three instances of Riboviria viruses from NCBI (Betacoronavirus RaTG13, bat-SL-CoVZC45, and bat-SL-CoVZXC21).

negative: Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests Summary Objectives A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Methods Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Results Two (2.1%) animal handlers had IF antibody titer of ≥1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Conclusions Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC.

negative: Phylogenetics, virus evolution and molecular epidemiology

negative: Structural analysis of SARS-CoV-2 and prediction of the human interactome Specific elements of viral genomes regulate interactions within host cells. Here, we calculated the secondary structure content of>2500 coronaviruses and computed>100000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We found that the 3 and 5 prime ends are the most structured elements in the viral genome and the 5 prime end has the strongest propensity to associate with human proteins. The domain encompassing nucleotides 23000-24000 is highly conserved both at the sequence and structural level, while the region upstream varies significantly. These two sequences code for a domain of the viral protein Spike S that interacts with the human receptor angiotensin-converting enzyme 2 (ACE2) and has the potential to bind sialic acids. Our predictions indicate that the first 1000 nucleotides in the 5 prime end can interact with proteins involved in viral RNA processing such as double-stranded RNA specific editases and ATP-dependent RNA-helicases, in addition to other high-confidence candidate partners. These interactions, previously reported to be also implicated in HIV, reveal important information on host-virus interactions. The list of transcriptional and post-transcriptional elements recruited by SARS-CoV-2 genome provides clues on the biological pathways associated with gene expression changes in human cells.

negative: Molecular epidemiological survey and phylogenetic analysis of bovine respiratory coronavirus in Japan from 2016 to 2018 Bovine coronavirus (BCoV) is an etiological agent of bovine respiratory disease (BRD). BRD is a costly illness worldwide; thus, epidemiological surveys of BCoV are important. Here, we conducted a molecular epidemiological survey of BCoV in respiratory-diseased and healthy cattle in Japan from 2016 to 2018. We found that 21.2% (58/273) of the respiratory-diseased cattle were infected with BCoV. The respiratory-diseased cattle had virus amounts 4.7 times higher than those in the asymptomatic cattle. Phylogenetic analyses showed that the BCoV identified in Japan after 2005 formed an individual lineage that was distinct from the strains found in other countries. These results suggest that BCoV is epidemic and has evolved uniquely in Japan.

negative: Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.

negative: Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2 Coronavirus disease 2019 (COVID-19) is newly emerging human infectious diseases, which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within two months of the outbreak, more than 80,000 cases of COVID-19 have been confirmed worldwide. Since the human to human transmission occurred easily and the human infection is rapidly increasing, the sensitive and early diagnosis is essential to prevent the global outbreak. Recently, World Health Organization (WHO) announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA. In this study, we compared the ability to detect SARS-CoV-2 RNA among the seven primer-probe sets for N gene and the three primer-probe sets for Orf1 gene. The result of the comparative analysis represented that the ‘2019-nCoV_N2, N3’ of USA and the ‘ORF1ab’ of China are the most sensitive primer-probe sets for N and Orf1 genes, respectively. Therefore, the appropriate combination from ORF1ab (China), 2019-nCoV_N2, N3 (USA), and NIID_2019-nCOV_N (Japan) sets should be selected for the sensitive and reliable laboratory confirmation of SARS-CoV-2.

negative: State-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in SARS-CoV-2 INTRODUCTION: The extreme health and economic problems in the world due to the SARS-CoV-2 infection have led to an urgent need to identify potential drug targets for treating coronavirus disease 2019 (COVID-19). The present state-of-the-art tool-based screening was targeted to identify drug targets among clinically approved drugs by uncovering SARS-CoV-2 helicase inhibitors through molecular docking analysis. MATERIAL AND METHODS: Helicase is a vital viral replication enzyme, which unwinds nucleic acids and separates the double-stranded nucleic acids into single-stranded nucleic acids. Hence, the SARS-CoV-2 helicase protein 3D structure was predicted, validated, and used to screen the druggable targets among clinically approved drugs such as protease inhibitor, nucleoside reverse transcriptase inhibitor, and non-nucleoside reverse transcriptase inhibitors, used to treat HIV infection using molecular docking analysis. RESULTS: Interaction with SARS-CoV-2 helicase, approved drugs, vapreotide (affinity: –12.88; S score: –9.84 kcal/mol), and atazanavir (affinity: –11.28; S score: –9.32 kcal/mol), approved drugs for treating AIDS-related diarrhoea and HIV infection, respectively, are observed with significantly low binding affinity and MOE score or binding free energy. The functional binding pockets of the clinically approved drugs on SARS-CoV-2 helicase protein molecule suggest that vapreotide and atazanavir may interrupt the activities of the SARS-CoV-2 helicase. CONCLUSIONS: The study suggests that vapreotide may be a choice of drug for wet lab studies to inhibit the infection of SARS-CoV-2.

negative: Towards a new paradigm linking virus molecular evolution and pathogenesis: experimental design and phylodynamic inference. Phylogenetic analysis has become a powerful tool for the investigation of evolution at a molecular level. During the last three decades, statistical phylogenetics has increasingly been applied to the study of microbial pathogens. The new field of phylodynamics was formally introduced in 2004 and encompasses the interaction between evolutionary and ecological processes that shape the spatiotemporal and phylogenetic patterns of infectious disease dynamics. This novel framework has significantly enhanced the study of measurable evolving pathogen populations, in particular RNA viruses and retroviruses. One of the major challenges in phylodynamic studies, however, is the generation of data in the form of dense coverage in sequence sampling coupled with high quality epidemiological and/or accurate clinical information. This review focuses specifically on experimental and data assembling strategies that are required to test multi-level phylodynamic hypotheses, ranging from intra-host viral evolution to population dynamics of infectious disease pandemics. Ultimately, bridging the gap between rational experimental design and phylodynamic inference will prove to be essential to take full advantage of this new exciting area of research.

negative: Is SARS-CoV-2 originated from laboratory? A rebuttal to the claim of formation via laboratory recombination

negative: Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

negative: Initial data from the COVID-19 Global Rheumatology Alliance provider registries – Authors' reply

negative: Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKβ and IFNG as upstream regulators of the transcriptional profile of ACE2(+)TMPRSS2(+) cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.

negative: Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

negative: Phylogenetic evidence for a mild H1 pandemic in the early 1900s.

negative: Differential Diagnosis of Common Acute Toxicologic Versus Nontoxicologic Illness This table outlines common toxicologic versus nontoxicologic rule outs based on clinical abnormalities seen in an acutely ill animal. The purpose is to provide an initial guideline for considering toxicologic versus nontoxicologic rule outs when a patient is presented to a practicing veterinarian. Major clinical abnormalities followed by common toxicologic rule outs and non-toxicologic rule outs have been listed so that practicing veterinarians can narrow down an etiology quickly. Based on history, physical examination findings, and blood work changes, once a reasonable etiology has been narrowed down or established, the reader is encouraged to review a more detailed discussion on management of the particular poisoning or disease listed in this or other references.

negative: Genomic analysis of Escherichia coli strains isolated from diseased chicken in the Czech Republic BACKGROUND: Avian pathogenic Escherichia coli (APEC) can cause various extraintestinal infections in poultry, resulting in massive economic losses in poultry industry. In addition, some avian E. coli strains may have zoonotic potential, making poultry a possible source of infection for humans. Due to its extreme genetic diversity, this pathotype remains poorly defined. This study aimed to investigate the diversity of colibacillosis-associated E. coli isolates from Central European countries with a focus on the Czech Republic. RESULTS: Of 95 clinical isolates subjected to preliminary characterization, 32 were selected for whole-genome sequencing. A multi resistant phenotype was detected in a majority of the sequenced strains with the predominant resistance to β-lactams and quinolones being associated with TEM-type beta-lactamase genes and chromosomal gyrA mutations respectively. The phylogenetic analysis confirmed a great diversity of isolates, that were derived from nearly all phylogenetic groups, with predominace of B2, B1 and C phylogroups. Clusters of closely related isolates within ST23 (phylogroup C) and ST429 (phylogroup B2) indicated a possible local spread of these clones. Besides, the ST429 cluster carried bla(CMY-2, − 59) genes for AmpC beta-lactamase and isolates of both clusters were generally well-equipped with virulence-associated genes, with considerable differences in distribution of certain virulence-associated genes between phylogenetically distant lineages. Other important and potentially zoonotic APEC STs were detected, incl. ST117, ST354 and ST95, showing several molecular features typical for human ExPEC. CONCLUSIONS: The results support the concept of local spread of virulent APEC clones, as well as of zoonotic potential of specific poultry-associated lineages, and highlight the need to investigate the possible source of these pathogenic strains.

negative: Genomic Sequencing and Analysis of Eight Camel-Derived Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Isolates in Saudi Arabia Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.

negative: Chapter 13 Viral Disease Abstract This chapter discusses infections of rats with viruses in the following 14 virus families: Adenoviridae, Arenaviridae, Coronaviridae, Flaviviridae, Hantaviridae, Hepeviridae, Herpesviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, and Reoviridae. Serological surveys indicate that parvoviruses, coronaviruses, cardioviruses, and pneumoviruses are the most prevalent in laboratory rats. A new polyomavirus and a new cardiovirus that cause disease in laboratory rats are described. Metagenomic analyses of feces or intestinal contents from wild rats have detected viruses from an additional nine virus families that could potentially cause infections in laboratory rats.

negative: A data-driven hypothesis on the epigenetic dysregulation of host metabolism by SARS coronaviral infection: potential implications for the SARS-CoV-2 modus operandi COVID-19, the disease caused by the novel SARS-CoV-2, a betacoronavirus structurally similar to SARS-CoV. Based on both structural and syndromic similarities with SARS-CoV, a hypothesis is formed on SARS-CoV-2 potential to affect the host’s metabolism as part of its lifecycle. This hypothesis is evaluated by (a) exploratory analysis of SARS-CoV / human transcriptomic interaction data and gene set enrichment analysis (b) a confirmatory, focused review of the literature based on the findings by (a). A STRING Viruses (available search for human – SARS-CoV (NCBI taxonomy Id: 9606 vs. NCBI taxonomy Id: 694009) genomic interactions reveals ten human proteins, interacting with SARS-CoV: SGTA, FGL2, SPECC1, STAT3, PHB, BCL2L1, PPP1CA, CAV1, JUN, XPO1. Gene set enrichment analyses (GSEA) with STRING on this network revealed their role as a putative protein – protein interaction network (PPI; Enrichment p-value=0.0296) mediating, viral parasitism, interleukin as well as insulin signaling, diabetes and triglyceride catabolism. In the literature, SARS-CoV has been known to cause de novo diabetes by ACE2-dependent uptake on pancreatic isle cells, and furthermore dysregulate lipid autophagy in favor of the viral lifecycle. Conversely, currently there are only non-causative, observational evidence of worse outcomes for COVID-19 patients with comorbid diabetes or hyperglycemia. No study has reported on the lipid profiles of COVID-19 patients; however, lipid-targeting molecules have been proposed as agents against SARS-CoV-2. Future studies, reporting on lipid and glucose metabolism of COVID-19 patients could help elucidate the disease’s seculae and aid drug design.

negative: A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption(1,2). There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity- purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

negative: Use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-nCoV: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target A novel coronavirus recently identified in Wuhan, China (SARS-CoV-2) has expanded the number of highly pathogenic coronaviruses affecting humans. The SARS-CoV-2 represents a potential epidemic or pandemic threat, which requires a quick response for preparedness against this infection. The present report uses the informational spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. The performed in silico analysis indicates that the newly emerging SARS-CoV-2 is closely related to severe acute respiratory syndrome (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) might be a putative receptor for the novel virus as well. Actin protein was also suggested as a host factor that participates in cell entry and pathogenesis of SARS-CoV-2; therefore, drugs modulating biological activity of this protein (e.g. ibuprofen) were suggested as potential candidates for treatment of this viral infection. Additional results indicated that civets and poultry are potential candidates for the natural reservoir of the SARS-CoV-2, and that domain 288-330 of S1 protein from the SARS-CoV-2 represents promising therapeutic and/or vaccine target.

negative: A Novel Construction of Genome Space with Biological Geometry A genome space is a moduli space of genomes. In this space, each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Currently, there is no method to represent genomes by a point in a space without losing biological information. Here, we propose a new graphical representation for DNA sequences. The breakthrough of the subject is that we can construct the moment vectors from DNA sequences using this new graphical method and prove that the correspondence between moment vectors and DNA sequences is one-to-one. Using these moment vectors, we have constructed a novel genome space as a subspace in R(N). It allows us to show that the SARS-CoV is most closely related to a coronavirus from the palm civet not from a bird as initially suspected, and the newly discovered human coronavirus HCoV-HKU1 is more closely related to SARS than to any other known member of group 2 coronavirus. Furthermore, we reconstructed the phylogenetic tree for 34 lentiviruses (including human immunodeficiency virus) based on their whole genome sequences. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships.

negative: Nosocomial outbreak of the Middle East Respiratory Syndrome coronavirus: A phylogenetic, epidemiological, clinical and infection control analysis BACKGROUND: Middle East Respiratory Syndrome coronavirus (MERS-CoV) continues to cause intermittent community and nosocomial outbreaks. Obtaining data on specific source(s) and transmission dynamics of MERS-CoV during nosocomial outbreaks has been challenging. We performed a clinical, epidemiological and phylogenetic investigation of an outbreak of MERS-CoV at a University Hospital in Riyadh, Kingdom of Saudi Arabia. METHODS: Clinical, epidemiological and infection control data were obtained from patients and Healthcare workers (HCWs). Full genome sequencing was conducted on nucleic acid extracted directly from MERS-CoV PCR-confirmed clinical samples and phylogenetic analysis performed. Phylogenetic analysis combined with published MERS-CoV genomes was performed. HCWs compliance with infection control practices was also assessed. RESULTS: Of 235 persons investigated, there were 23 laboratory confirmed MERS cases, 10 were inpatients and 13 HCWs. Eight of 10 MERS inpatients died (80% mortality). There were no deaths among HCWs. The primary index case assumed from epidemiological investigation was not substantiated phylogenetically. 17/18 of MERS cases were linked both phylogenetically and epidemiologically. One asymptomatic HCW yielded a MERS-CoV genome not directly linked to any other case in the investigation. Five HCWs with mild symptoms yielded >75% full MERS-CoV genome sequences. HCW compliance with use of gowns was 62.1%, gloves 69.7%, and masks 57.6%. CONCLUSIONS: Several factors and sources, including a HCW MERS-CoV ‘carrier phenomenon’, occur during nosocomial MERS-CoV outbreaks. Phylogenetic analyses of MERS-CoV linked to clinical and epidemiological information is essential for outbreak investigation. The specific role of apparently healthy HCWs in causing nosocomial outbreaks requires further definition.

negative: Patient-derived mutations impact pathogenicity of SARS-CoV-2 The sudden outbreak of the severe acute respiratory syndrome-coronavirus (SARS-CoV-2) has spread globally with more than 1,300,000 patients diagnosed and a death toll of 70,000. Current genomic survey data suggest that single nucleotide variants (SNVs) are abundant. However, no mutation has been directly linked with functional changes in viral pathogenicity. Here we report functional characterizations of 11 patient-derived viral isolates, all of which have at least one mutation. Importantly, these viral isolates show significant variation in cytopathic effects and viral load, up to 270-fold differences, when infecting Vero-E6 cells. We observed intrapersonal variation and 6 different mutations in the spike glycoprotein (S protein), including 2 different SNVs that led to the same missense mutation. Therefore, we provide direct evidence that the SARS-CoV-2 has acquired mutations capable of substantially changing its pathogenicity.

negative: Unusual Influenza A Viruses in Bats Influenza A viruses infect a remarkably diverse number of hosts. Two completely new influenza A virus subtypes were recently discovered in bats, dramatically expanding the host range of the virus. These bat viruses are extremely divergent from all other known strains and likely have unique replication cycles. Phylogenetic analysis indicates long-term, isolated evolution in bats. This is supported by a high seroprevalence in sampled bat populations. As bats represent ~20% of all classified mammals, these findings suggests the presence of a massive cryptic reservoir of poorly characterized influenza A viruses. Here, we review the exciting progress made on understanding these newly discovered viruses, and discuss their zoonotic potential.

negative: Retinal pathology in multiple sclerosis: insight into the mechanisms of neuronal pathology

negative: Molecular biological analysis of genotyping and phylogeny of severe acute respiratory syndrome associated coronavirus. BACKGROUND SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS) which has been associated with outbreaks of SARS in Guangdong, Hong Kong and Beijing of China, and other regions worldwide. SARS-CoV from human has shown some variations but its origin is still unknown. The genotyping and phylogeny of SARS-CoV were analyzed and reported in this paper. METHODS Full or partial genomes of 44 SARS-CoV strains were collected from GenBank. The genotype, single nucleotide polymorphism and phylogeny of these SARS-CoV strains were analyzed by molecular biological, bioinformatic and epidemiological methods. RESULTS There were 188 point mutations in the 33 virus full genomes with the counts of mutation mounting to 297. Further analysis was carried out among 36 of 188 loci with more than two times of mutation. All the 36 mutation loci occurred in coding sequences and 22 loci were non-synonymous. The gene mutation rates of replicase 1AB, S2 domain of spike glycoprotein and nucleocapsid protein were lower (0.079% - 0.103%). There were 4 mutation loci in S1 domain of spike glycoprotein. The gene mutation rate of ORF10 was the highest (3.333%) with 4 mutation loci in this small domain (120 bp) and 3 of 4 loci related to deletion mutation. By bioinformatics processing and analysis, the nucleotides at 7 loci of genome (T:T:A:G:T:C:T/C:G:G:A:C:T:C) can classify SARS-CoV into two types. Therefore a novel definition is put forward that according to these 7 loci of mutation, 40 strains of SARS-CoV in GenBank can be grouped into two genotypes, T:T:A:G:T:C:T and C:G:G:A:C:T:C, and named as SARS-CoV Yexin genotype and Xiaohong genotype. The two genotypes can be further divided into some sub-genotypes. These genotypes can also be approved by phylogenetic tree of three levels of 44 loci of mutation, spike glycoprotein gene and complete genome sequence. Compared to various strains among SARS-CoV Yexin genotype and Xiaohong genotype, GD01 strain of Yexin genotype is more closely related to SARS-CoV like-virus from animals. CONCLUSION The results mentioned above suggest that SARS-CoV is responding to host immunological pressures and experiencing variation which provide clues, information and evidence of molecular biology for the clinical pathology, vaccine developing and epidemic investigation.

negative: Dealing with a pandemic: the Kerala Model of containment strategy for COVID-19.

negative: Protein Structure and Sequence Reanalysis of 2019-nCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1 [Image: see text] As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, the careful analysis of its transmission and cellular mechanisms is sorely needed. In this Communication, we first analyzed two recent studies that concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions share a unique similarity to HIV-1. However, the reimplementation of the analyses, built on larger scale data sets using state-of-the-art bioinformatics methods and databases, presents clear evidence that rebuts these conclusions. Next, using metagenomic samples from Manis javanica, we assembled a draft genome of the 2019-nCoV-like coronavirus, which shows 73% coverage and 91% sequence identity to the 2019-nCoV genome. In particular, the alignments of the spike surface glycoprotein receptor binding domain revealed four times more variations in the bat coronavirus RaTG13 than in the Manis coronavirus compared with 2019-nCoV, suggesting the pangolin as a missing link in the transmission of 2019-nCoV from bats to human.

negative: Regional anesthesia in patients with suspected COVID-19 infection

negative: Bugs in the System Immunity to respiratory virus infection is governed by complex biological networks that influence disease progression and pathogenesis. Systems biology provides an opportunity to explore and understand these multifaceted interactions based on integration and modeling of multiple biological parameters. In this review, we describe new and refined systems-based approaches used to model, identify, and validate novel targets within complex networks following influenza and coronavirus infection. In addition, we propose avenues for extension and expansion that can revolutionize our understanding of infectious disease processes. Together, we hope to provide a window into the unique and expansive opportunity presented by systems biology to understand complex disease processes within the context of infectious diseases.

negative: Phylogenetic analysis of human coronavirus NL63 circulating in Italy Abstract Background Five known human coronaviruses infect the human respiratory tract: HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63 and HCoV-HKU1. Objectives To evaluate the prevalence of HCoV-NL63 in hospitalized adult patients and to perform molecular characterization of Italian strains. Study Design HCoV-NL63 was sought by RT-PCR in 510 consecutive lower respiratory tract (LRT) samples, collected from 433 Central-Southern Italy patients over a 1-year period. Phylogenetic analysis was performed by partial sequencing of S and ORF1a. Additional S sequences from Northern Italy were included in the phylogenetic trees. Results HCoV-NL63 was detected in 10 patients (2.0%) with symptomatic respiratory diseases, mainly during winter. Phylogenetic analysis indicated a certain degree of heterogeneity in Italian isolates. The ORF1a gene clustering in phylogenetic trees did not match with that of the S gene. Conclusions As observed by others, HCoV-NL63 is often associated with another virus. Phylogenetic characterization of HCoV-NL63 circulating in Italy indicates that this virus circulates as a mixture of variant strains, as observed in other countries.

negative: The current understanding and potential therapeutic options to combat COVID-19 Abstract The ongoing wreaking global outbreak of the novel human beta coronavirus (CoV) pathogen was presumed to be from a seafood wholesale market in Wuhan, China, belongs to the Coronaviridae family in the Nidovirales order. The virus is highly contagious with potential human-human transmission which was named as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread across six continents and emerged as a global pandemic in short span with alarming levels of spread and severity. This virus associated symptoms and infectious respiratory illness is designated as coronavirus disease 19 (COVID-19). The SARS-CoV-2 possesses enveloped club-like spike protein projections with positive-sense large RNA genome and has a unique replication strategy. This virus was believed to have zoonotic origin with genetical identity to bat and pangolin CoV. In the current review, we introduce a general overview about the human CoVs and the associated diseases, the origin, structure, replication and key clinical events that occur in the COVID-19 pathogenicity. Furthermore, we focused on possible therapeutic options such as repurposing drugs including antimalarials, antivirals, antiparasitic drugs, and anti-HIV drugs, as well as monoclonal antibodies, vaccines as potential treatment options. Also we have summarized the latest research progress on the usage of stem cell therapy, human convalescent serum, interferon's, in the treatment of COVID-19.

negative: The establishment of reference sequence for SARS‐CoV‐2 and variation analysis Starting around December 2019, an epidemic of pneumonia, which was named COVID‐19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full‐length genomic sequences of SARAS‐CoV‐2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS‐CoV‐2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%‐100%) at the nucleotide level and 99.99% (99.79%‐100%) at the amino acid level. Although overall variation in open‐reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS‐COV‐2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS‐CoV‐2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS‐CoV‐2 infection in the future.

negative: Evidence and possible mechanisms of rare maternal-fetal transmission of SARS-CoV-2. While SARS-CoV-2 infection has spread rapidly worldwide, data remains scarce about the natural history of infection in pregnant women and the risk of mother-to-fetal transmission. Current data indicates that viral RNA levels in maternal blood are low and there is no evidence of placental infection with SARS-CoV-2. Published reports to date suggest that perinatal transmission of SARSCoV- 2 can occur but is rare. Among 179 newborns tested for SARS-CoV2 at birth from mothers with COVID-19, transmission was suspected in 8 cases, 5 with positive nasopharyngeal SARS-CoV-2 RT-PCR and 3 with SARS-CoV-2 IgM. However, these cases arise from maternal infection close to childbirth and there are no information about exposition during first or second trimester of pregnancy. Welldesigned prospective cohort studies with rigorous judgement criteria are needed to determine the incidence and risk factors for perinatal transmission of SARS-CoV-2.

negative: METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies - Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses We present METATRYP version-2 software that identifies shared peptides across organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include: ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the Least Common Ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine database confirms low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed for ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A Coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa, sharing 0.1% peptides or less (1 peptide) with the Influenza A & B pan-proteomes, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches. Statement of significance When assigning taxonomic attribution in bottom-up metaproteomics, the potential for shared tryptic peptides among organisms in mixed communities should be considered. The software program METATRYP v 2 and associated interactive web portals enables users to identify the frequency of shared tryptic peptides among taxonomic groups and evaluate the occurrence of specific tryptic peptides within complex communities. METATRYP facilitates phyloproteomic studies of taxonomic groups and supports the identification and evaluation of potential metaproteomic biomarkers.

negative: Structure of replicating SARS-CoV-2 polymerase The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’ that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19).

negative: Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration with high mortality rates in swine. It has become increasingly problematic in China. Since the nucleocapsid (N) protein is highly conserved, it is a candidate protein for early diagnosis and vaccine development. In this study, the N genes of 15 PEDV strains were amplified by RT-PCR and cloned into the pMT-19T vector, sequenced, and compared to each other as well as to PEDV reference strains. The nucleotide sequences of the N gene of the Chinese PEDV strains consist of 1326 nucleotides and encode a 441-aa-long peptide. The nucleotide sequences of the fifteen PEDV strains in our study were 96.1-100 % identical to each other, and the deduced amino acid sequences were 94.8-100 % identical. Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.2-99.7 % identical to those of the Chinese PEDV strains, and their deduced amino acid sequences were 94.1-99.5 % identical. In addition, the fifteen strains showed a high degree of nucleotide sequence identity to the early domestic strains (98.4-99.7 %) except the LZC strain, but less sequence identity to the vaccine strain (CV777) used in China (94.7-97.7 %). Phylogenetic analysis showed that the Chinese PEDV strains are composed of a separate cluster including three early domestic strains (JS-2004-02, LJB/03 and DX) but differ genetically from the vaccine strain (CV777) and the early Korean strains (Chinju99 and SM98).

negative: Genome Sequencing of a Severe Acute Respiratory Syndrome Coronavirus 2 Isolate Obtained from a South African Patient with Coronavirus Disease 2019 As a contribution to the global efforts to track and trace the ongoing coronavirus pandemic, here we present the sequence, phylogenetic analysis, and modeling of nonsynonymous mutations for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome that was detected in a South African patient with coronavirus disease 2019 (COVID-19).

negative: Analysis of SARS-CoV-2 RNA-Sequences by Interpretable Machine Learning Models We present an approach to investigate SARS-CoV-2 virus sequences based on alignment-free methods for RNA sequence comparison. In particular, we verify a given clustering result for the GISAID data set, which was obtained analyzing the molecular differences in coronavirus populations by phylogenetic trees. For this purpose, we use alignment-free dissimilarity measures for sequences and combine them with learning vector quantization classifiers for virus type discriminant analysis and classification. Those vector quantizers belong to the class of interpretable machine learning methods, which, on the one hand side provide additional knowledge about the classification decisions like discriminant feature correlations, and on the other hand can be equipped with a reject option. This option gives the model the property of self controlled evidence if applied to new data, i.e. the models refuses to make a classification decision, if the model evidence for the presented data is not given. After training such a classifier for the GISAID data set, we apply the obtained classifier model to another but unlabeled SARS-CoV-2 virus data set. On the one hand side, this allows us to assign new sequences to already known virus types and, on the other hand, the rejected sequences allow speculations about new virus types with respect to nucleotide base mutations in the viral sequences. Author summary The currently emerging global disease COVID-19 caused by novel SARS-CoV-2 viruses requires all scientific effort to investigate the development of the viral epidemy, the properties of the virus and its types. Investigations of the virus sequence are of special interest. Frequently, those are based on mathematical/statistical analysis. However, machine learning methods represent a promising alternative, if one focuses on interpretable models, i.e. those that do not act as black-boxes. Doing so, we apply variants of Learning Vector Quantizers to analyze the SARS-CoV-2 sequences. We encoded the sequences and compared them in their numerical representations to avoid the computationally costly comparison based on sequence alignments. Our resulting model is interpretable, robust, efficient, and has a self-controlling mechanism regarding the applicability to data. This framework was applied to two data sets concerning SARS-CoV-2. We were able to verify previously published virus type findings for one of the data sets by training our model to accurately identify the virus type of sequences. For sequences without virus type information (second data set), our trained model can predict them. Thereby, we observe a new scattered spreading of the sequences in the data space which probably is caused by mutations in the viral sequences.

negative: Epidemiology and phylogenetic analysis of respiratory viruses from 2012 to 2015 – A sentinel surveillance report from union territory of Puducherry, India Abstract Background Acute respiratory infections (ARI) are the most common illnesses affecting people of all ages worldwide. Viruses contribute to 30–70% of acute respiratory infections. Identification of these respiratory viruses is not given high priority except influenza; however, the knowledge about prevalence of non-influenza viruses, their seasonal pattern and genetic evolution have significant epidemiological value. Methods As a part of National Influenza-like illness surveillance programme, respiratory specimens were collected children and adults with symptoms of ILI or ARI, between January 2012 and March 2015 (including SARI cases). Real-time PCR was done to identify 13 respiratory viruses. Sequencing was done for representative isolates of each virus using ABI 3730 Genetic Analyzer. Results During the study period between January 2012 and March 2015, a total of 648 patients with symptoms of ARI were included in this study. The mean age of the patients was 20.2 years (SD = 19.13, median = 18); 292 (45.1%) were children (≤13 years) and 356 (54.9%) were adults. Respiratory viruses were identified in 44% (287/648) of all patients. Influenza accounted for the maximum number of cases- 179/648 (27.6%). Among the non-influenza viruses, RSV predominated with 34 cases (5.2%), followed by HMPV 24 (3.7%) and PIV-3 20 (3%). Four patients died due to INF A/H1N1 as a result of acute respiratory distress syndrome (ARDS) (CFR 3.7%). Among the non-influenza viruses, no particular seasonality pattern was observed over the different months of the study period. Conclusion Antibiotic usage in treating acute respiratory infections empirically is not justified as nearly half of ARI are due to viruses; nearly 28% of them were due to influenza viruses. Among the non-influenza viruses, RSV predominated, followed by HMPV. This study is based on an active influenza surveillance initiated after 2009 pandemic influenza outbreak, in the Union territory of Puducherry which has contributed significantly to the knowledge of the burden of influenza and non-influenza viruses among children and adults. Such surveillance network has paved the way for better diagnosis and timely therapeutic interventions.

negative: The potential effects of widespread community transmission of SARS-CoV-2 infection in the World Health Organization African Region: a predictive model The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been unprecedented in its speed and effects. Interruption of its transmission to prevent widespread community transmission is critical because its effects go beyond the number of COVID-19 cases and deaths and affect the health system capacity to provide other essential services. Highlighting the implications of such a situation, the predictions presented here are derived using a Markov chain model, with the transition states and country specific probabilities derived based on currently available knowledge. A risk of exposure, and vulnerability index are used to make the probabilities country specific. The results predict a high risk of exposure in states of small size, together with Algeria, South Africa and Cameroon. Nigeria will have the largest number of infections, followed by Algeria and South Africa. Mauritania would have the fewest cases, followed by Seychelles and Eritrea. Per capita, Mauritius, Seychelles and Equatorial Guinea would have the highest proportion of their population affected, while Niger, Mauritania and Chad would have the lowest. Of the World Health Organization's 1 billion population in Africa, 22% (16%–26%) will be infected in the first year, with 37 (29 – 44) million symptomatic cases and 150 078 (82 735–189 579) deaths. There will be an estimated 4.6 (3.6–5.5) million COVID-19 hospitalisations, of which 139 521 (81 876–167 044) would be severe cases requiring oxygen, and 89 043 (52 253–106 599) critical cases requiring breathing support. The needed mitigation measures would significantly strain health system capacities, particularly for secondary and tertiary services, while many cases may pass undetected in primary care facilities due to weak diagnostic capacity and non-specific symptoms. The effect of avoiding widespread and sustained community transmission of SARS-CoV-2 is significant, and most likely outweighs any costs of preventing such a scenario. Effective containment measures should be promoted in all countries to best manage the COVID-19 pandemic.

negative: Molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus HX strain isolated from China BACKGROUND: Porcine transmissible gastroenteritis virus (TGEV) is the major etiological agent of viral enteritis and severe diarrhea in suckling piglets. In China, TGEV has caused great economic losses, but its role in epidemic diarrhea is unclear. This study aims to reveal the etiological role of TGEV in piglet diarrhea via molecular characterization and phylogenetic analysis. RESULTS: A TGEV-HX strain was isolated from China, and its complete genome was amplified, cloned, and sequenced. Sequence analysis indicated that it was conserved in the 5′ and 3′-non-translated regions, and there were no insertions or deletions in nonstructural genes, such as ORF1a, ORF1b, ORF3a, ORF3b, and ORF7, as well as in genes encoding structural proteins, such as the envelope (E), membrane (M), and nucleoprotein (N) proteins. Furthermore, the phylogenetic analysis indicated that the TGEV-HX strain was more similar to the TGEV Purdue cluster than to the Miller cluster. CONCLUSIONS: The present study described the isolation and genetic characterization of a TGEV-HX strain. The detailed analysis of the genetic variation of TGEVs in China provides essential information for further understanding the evolution of TGEVs.

negative: From SARS and MERS CoVs to SARS‐CoV‐2: Moving toward more biased codon usage in viral structural and nonstructural genes BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an emerging disease with fatal outcomes. In this study, a fundamental knowledge gap question is to be resolved by evaluating the differences in biological and pathogenic aspects of SARS‐CoV‐2 and the changes in SARS‐CoV‐2 in comparison with the two prior major COV epidemics, SARS and Middle East respiratory syndrome (MERS) coronaviruses. METHODS: The genome composition, nucleotide analysis, codon usage indices, relative synonymous codons usage, and effective number of codons (ENc) were analyzed in the four structural genes; Spike (S), Envelope (E), membrane (M), and Nucleocapsid (N) genes, and two of the most important nonstructural genes comprising RNA‐dependent RNA polymerase and main protease (Mpro) of SARS‐CoV‐2, Beta‐CoV from pangolins, bat SARS, MERS, and SARS CoVs. RESULTS: SARS‐CoV‐2 prefers pyrimidine rich codons to purines. Most high‐frequency codons were ending with A or T, while the low frequency and rare codons were ending with G or C. SARS‐CoV‐2 structural proteins showed 5 to 20 lower ENc values, compared with SARS, bat SARS, and MERS CoVs. This implies higher codon bias and higher gene expression efficiency of SARS‐CoV‐2 structural proteins. SARS‐CoV‐2 encoded the highest number of over‐biased and negatively biased codons. Pangolin Beta‐CoV showed little differences with SARS‐CoV‐2 ENc values, compared with SARS, bat SARS, and MERS CoV. CONCLUSION: Extreme bias and lower ENc values of SARS‐CoV‐2, especially in Spike, Envelope, and Mpro genes, are suggestive for higher gene expression efficiency, compared with SARS, bat SARS, and MERS CoVs.

negative: Unsupervised cluster analysis of SARS-CoV-2 genomes indicates that recent (June 2020) cases in Beijing are from a genetic subgroup that consists of mostly European and South(east) Asian samples, of which the latter are the most recent Research efforts of the ongoing SARS-CoV-2 pandemic have focused on viral genome sequence analysis to understand how the virus spread across the globe. Here, we assess three recently identified SARS-CoV-2 genomes in Beijing from June 2020 and attempt to determine the origin of these genomes, made available in the GISAID database. The database contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Including the three new samples and excluding samples with missing annotations, we analyzed 7, 643 SARS-CoV-2 genomes. Using principal component analysis computed on a similarity matrix that compares all pairs of the SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index, we find that the newly discovered virus genomes from Beijing are in a genetic cluster that consists mostly of cases from Europe and South(east) Asia. The sequences of the new cases are most related to virus genomes from a small number of cases from China (March 2020), cases from Europe (February to early May 2020), and cases from South(east) Asia (May to June 2020). These findings could suggest that the original cases of this genetic cluster originated from China in March 2020 and were re-introduced to China by transmissions from samples from South(east) Asia between April and June 2020.

negative: Informational needs assessment of non‐Hodgkin lymphoma survivors and their physicians

negative: The coding capacity of SARS-CoV-2 SARS-CoV-2 is a coronavirus responsible for the COVID-19 pandemic. In order to understand its pathogenicity, antigenic potential and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 translated open reading frames (ORF)s in an unbiased and open-ended manner. Utilizing a suit of ribosome profiling techniques, we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral ORFs and to identify 23 novel unannotated viral ORFs. These ORFs include several in-frame internal ORFs lying within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. Finally, we detected a prominent initiation at a CUG codon located in the 5’UTR. Although this codon is shared by all SARS-CoV-2 transcripts, the initiation was specific to the genomic RNA, indicating that the genomic RNA harbors unique features that may affect ribosome engagement. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.

negative: Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2 Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV) and 2019 novel coronavirus (2019-nCoV, also known as SARS-CoV-2), lead global epidemics with high morbidity and mortality. However, there are currently no effective drugs targeting 2019-nCoV/SARS-CoV-2. Drug repurposing, representing as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. In this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the HCoV–host interactome and drug targets in the human protein–protein interaction network. Phylogenetic analyses of 15 HCoV whole genomes reveal that 2019-nCoV/SARS-CoV-2 shares the highest nucleotide sequence identity with SARS-CoV (79.7%). Specifically, the envelope and nucleocapsid proteins of 2019-nCoV/SARS-CoV-2 are two evolutionarily conserved regions, having the sequence identities of 96% and 89.6%, respectively, compared to SARS-CoV. Using network proximity analyses of drug targets and HCoV–host interactions in the human interactome, we prioritize 16 potential anti-HCoV repurposable drugs (e.g., melatonin, mercaptopurine, and sirolimus) that are further validated by enrichment analyses of drug-gene signatures and HCoV-induced transcriptomics data in human cell lines. We further identify three potential drug combinations (e.g., sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the “Complementary Exposure” pattern: the targets of the drugs both hit the HCoV–host subnetwork, but target separate neighborhoods in the human interactome network. In summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations targeting 2019-nCoV/SARS-CoV-2.

negative: Cardiac Magnetic Resonance characterization of myocarditis-like acute cardiac syndrome in COVID-19

negative: Evolutionary Dynamics of MERS-CoV: Potential Recombination, Positive Selection and Transmission Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to beta group of coronavirus and was first discovered in 2012. MERS-CoV can infect multiple host species and cause severe diseases in human. We conducted a series of phylogenetic and bioinformatic analyses to study the evolution dynamics of MERS-CoV among different host species with genomic data. Our analyses show: 1) 28 potential recombinant sequences were detected and they can be classified into seven potential recombinant types; 2) The spike (S) protein of MERS-CoV was under strong positive selection when MERS-CoV transmitted from their natural host to human; 3) Six out of nine positive selection sites detected in spike (S) protein are located in its receptor-binding domain which is in direct contact with host cells; 4) MERS-CoV frequently transmitted back and forth between human and camel after it had acquired the human-camel infection capability. Together, these results suggest that potential recombination events might have happened frequently during MERS-CoV’s evolutionary history and the positive selection sites in MERS-CoV’s S protein might enable it to infect human.

negative: The population genetics and evolutionary epidemiology of RNA viruses RNA viruses are ubiquitous intracellular parasites that are responsible for many emerging diseases, including AIDS and SARS. Here, we discuss the principal mechanisms of RNA virus evolution and highlight areas where future research is required. The rapidity of sequence change in RNA viruses means that they are useful experimental models for the study of evolution in general and it enables us to watch them change in 'real time', and retrace the spread through populations with molecular phylogenies. An understanding of the mechanisms of RNA virus sequence change is also crucial to predicting important aspects of their emergence and long-term evolution.

negative: The origin and underlying driving forces of the SARS-CoV-2 outbreak BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019–12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

negative: Dynamics of the ACE2 - SARS-CoV/SARS-CoV-2 spike protein interface reveal unique mechanisms The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major public health concern. A handful of static structures now provide molecular insights into how SARS-CoV-2 and SARS-CoV interact with its host target, which is the angiotensin converting enzyme 2 (ACE2). Molecular recognition, binding and function are dynamic processes. To evaluate this, multiple all atom molecular dynamics simulations of at least 500 ns each were performed to better understand the structural stability and interfacial interactions between the receptor binding domain of the spike protein of SARS-CoV-2 and SARS-CoV bound to ACE2. Several contacts were observed to form, break and reform in the interface during the simulations. Our results indicate that SARS-CoV and SARS-CoV-2 utilizes unique strategies to achieve stable binding to ACE2. Several differences were observed between the residues of SARS-CoV-2 and SARS-CoV that consistently interacted with ACE2. Notably, a stable salt bridge between Lys417 of SARS-CoV-2 spike protein and Asp30 of ACE2 as well as three stable hydrogen bonds between Tyr449, Gln493, and Gln498 of SARS-CoV-2 and Asp38, Glu35, and Lys353 of ACE2 were observed, which were absent in the SARS-CoV-ACE2 interface. Some previously reported residues, which were suggested to enhance the binding affinity of SARS-CoV-2, were not observed to form stable interactions in these simulations. Stable binding to the host receptor is crucial for virus entry. Therefore, special consideration should be given to these stable interactions while designing potential drugs and treatment modalities to target or disrupt this interface.

negative: Comparative analysis of non structural protein 1 of SARS-COV2 with SARS-COV1 and MERS-COV: An in silico study The recently emerged SARS-COV2 caused a major pandemic of coronavirus disease (COVID-19). The main goal of this study is to elucidate the structural conformations of non structural protein 1(nsp1), prediction of epitope sites and identification of important residues for targeted therapy against COVID-19. In this study, molecular modelling coupled with molecular dynamics simulations were performed to analyse the conformational change of SARS-COV1, SARS-COV2 and MERS-COV at molecular level. Free energy landscape was constructed by using the first (PC1) and second (PC2) principle components. From the sequence alignment it was observed when compared to SERS-COV1 28 mutations are present in SERS-COV2 nsp1 protein. Several B-cell and T-cell epitopes were identified by immunoinformatics study. The ΔG values for SARS-COV1, SARS-COV2 and MERS-COV nsp1 proteins were 4.44, 5.82 and 6.15 kJ/mol respectively. SARS-COV2 nsp1 protein binds with the interface region of the palm and finger domain of POLA1 by using hydrogen bonds and salt bridges interactions. The present study provided a comprehensive structural model of nsp1 by threading process. The MD simulation parameters indicated that all three nsp1 proteins were stable during the simulation run. These findings can be used to develop therapeutics specific against COVID-19. Highlights Structural elucidation at molecular level of nsp1 of SARS-COV1, SARS-COV2, and MERS-COV Identifications of epitopes by immunoinformatics approach SARS-COV2 nsp1 cover a large conformational space due to greater flexibility Molecular docking between SARS-COV2 nsp1 and POLA1 to identify important residues Structural insights of nsp1 could be used in drug design process against COVID-19 Graphical abstract

negative: Delivery Systems for the Direct Application of siRNAs to Induce RNA Interference (RNAi) In Vivo RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described.

negative: Clinical issues and research in respiratory failure from severe acute respiratory syndrome. The National Heart, Lung, and Blood Institute, along with the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases, convened a panel to develop recommendations for treatment, prevention, and research for respiratory failure from severe acute respiratory syndrome (SARS) and other newly emerging infections. The clinical and pathological features of acute lung injury (ALI) from SARS appear indistinguishable from ALI from other causes. The mainstay of treatments for ALI remains supportive. Patients with ALI from SARS who require mechanical ventilation should receive a lung protective, low tidal volume strategy. Adjuvant treatments recommended include prevention of venous thromboembolism, stress ulcer prophylaxis, and semirecumbent positioning during ventilation. Based on previous experience in Canada, infection control resources and protocols were recommended. Leadership structure, communication, training, and morale are an essential aspect of SARS management. A multicenter, placebo-controlled trial of corticosteroids for late SARS is justified because of widespread clinical use and uncertainties about relative risks and benefits. Studies of combined pathophysiologic endpoints were recommended, with mortality as a secondary endpoint. The group recommended preparation for studies, including protocols, ethical considerations, Web-based registries, and data entry systems.

negative: Phylogenetic analysis of bovine astrovirus in Korean cattle Bovine astrovirus (BAstV) belongs to a genetically divergent lineage within the genus Mamastrovirus. The present study showed that BAstV was associated with the gastroenteric tracts of cattle in nine positive fecal samples from 115 cattle, whereas no positive samples were found in the brain tissues of 14 downer cattle. Interestingly, the positive diarrheal samples were obtained mainly from calves aged 14 days–3 months. Bayesian inference tree analysis of the partial ORF1ab and capsid (ORF2) gene sequences of BAstVs identified four divergent groups. Eleven BAstVs, four porcine astroviruses, and two deer astroviruses (DAstVs; CcAstV-1 and -2) belonged to group 1; group 2 contained two BAstVs (BAstK08–51 and BAstK10–96) with another two in group 3 (BAstK08–2 and BAstK08–53); and group 4 comprised the BAstV-NeuroS1 strain derived from a cattle brain tissue sample and an ovine astrovirus. The same divergent groups were obtained when the pairwise alignments were produced using both amino acid and nucleotide sequences. The Korean BAstVs isolated from infected cattle had a nationwide distribution and they belonged to groups 1, 2, and 3. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-013-1013-0) contains supplementary material, which is available to authorized users.

negative: A Public Website for the Automated Assessment and Validation of SARS-CoV-2 Diagnostic PCR Assays Summary: Polymerase chain reaction-based assays are the current gold standard for detecting and diagnosing SARS-CoV-2. However, as SARS-CoV-2 mutates, we need to constantly assess whether existing PCR-based assays will continue to detect all known viral strains. To enable the continuous monitoring of SARS-CoV-2 assays, we have developed a web-based assay validation algorithm that checks existing PCR-based assays against the ever-expanding genome databases for SARS-CoV-2 using both thermodynamic and edit-distance metrics. The assay screening results are displayed as a heatmap, showing the number of mismatches between each detection and each SARS-CoV-2 genome sequence. Using a mismatch threshold to define detection failure, assay performance is summarized with the true positive rate (recall) to simplify assay comparisons. Availability: https://covid19.edgebioinformatics.org/#/assayValidation. Contact: Jason Gans (jgans@lanl.gov) and Patrick Chain (pchain@lanl.gov)

negative: What country, university or research institute, performed the best on COVID-19? Bibliometric analysis of scientific literature In this article, we conduct data mining to discover the countries, universities and companies, produced or collaborated the most research on Covid-19 since the pandemic started. We present some interesting findings, but despite analysing all available records on COVID-19 from the Web of Science Core Collection, we failed to reach any significant conclusions on how the world responded to the COVID-19 pandemic. Therefore, we increased our analysis to include all available data records on pandemics and epidemics from 1900 to 2020. We discover some interesting results on countries, universities and companies, that produced collaborated most the most in research on pandemic and epidemics. Then we compared the results with the analysing on COVID-19 data records. This has created some interesting findings that are explained and graphically visualised in the article.

negative: Identification of a common deletion in the spike protein of SARS-CoV-2 Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

negative: Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study BACKGROUND: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin. METHODS: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85–95%, and four 30–50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done. FINDINGS: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction. INTERPRETATION: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R(0) values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed. FUNDING: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

negative: Quasispecies composition and phylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats Quasispecies composition and tissue distribution of feline coronaviruses (FCoVs) were studied in naturally infected cats. The genomic complexity of FCoVs was investigated using single-strand conformational polymorphism (SSCP) analysis of N and ORF7b amplicons, and the evolutionary process was investigated by sequence-based phylogenetic analysis. SSCP analysis showed high heterogeneity of the FCoV genome which was correlated with the seriousness of the clinical form. The two genomic regions analysed showed different levels of variation; the N region demonstrated significant heterogeneity as compared to ORF7b. Phylogenetic analysis of the nucleotide sequences showed the clear separation of sequences analysed on the basis of virulence and geographical origin. A maximum likelihood analysis of N and ORF7b data sets showed a situation of strong heterogeneity for the N region.

negative: Molecular epidemiology and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea Porcine epidemic diarrhea virus (PEDV) has caused devastating enteric disease in Korean pig farms since its first identification in 1992 in Korea. In the present study, the molecular epidemiology, genetic diversity, and phylogenetic relationship of Korean PEDV field isolates to other reference strains were analyzed using the complete E gene. Genetic analysis showed that each PEDV group had several unique characteristics, which indicated that a specific group PEDVs may be differentiated from another group PEDVs. Phylogenetic analysis showed that recent prevalent Korean PEDV field isolates are closely related to the Chinese field strains and differ genetically from the European strains and the vaccine strains used in Korea, which raises questions of whether a new-type PEDV vaccine may be necessary for preventing PEDV infection more effectively in Korea. Notably, a large deletion identified only in the attenuated DR13 can be utilized as a genetic marker, and the methods developed in this study will help to rapidly detect and differentiate PEDVs.

negative: Coronavirus Genomics and Bioinformatics Analysis The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us an unprecedented opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Variable numbers of small ORFs are present between the various conserved genes (ORF1ab, spike, envelope, membrane and nucleocapsid) and downstream to nucleocapsid gene in different coronavirus lineages. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C and D, exist. A fourth genus, Deltacoronavirus, which includes bulbul coronavirus HKU11, thrush coronavirus HKU12 and munia coronavirus HKU13, is emerging. Molecular clock analysis using various gene loci revealed that the time of most recent common ancestor of human/civet SARS related coronavirus to be 1999–2002, with estimated substitution rate of 4×10(−4) to 2×10(−2) substitutions per site per year. Recombination in coronaviruses was most notable between different strains of murine hepatitis virus (MHV), between different strains of infectious bronchitis virus, between MHV and bovine coronavirus, between feline coronavirus (FCoV) type I and canine coronavirus generating FCoV type II, and between the three genotypes of human coronavirus HKU1 (HCoV-HKU1). Codon usage bias in coronaviruses were observed, with HCoV-HKU1 showing the most extreme bias, and cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape such codon usage bias in coronaviruses.

negative: Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China To investigate bat coronaviruses (CoVs), we collected 132 rectal swabs and urine samples from five bat species in three countries in southwestern China. Seven CoVs belonging to distinct groups of severe acute respiratory syndrome (SARS)-like CoVs and α-CoVs were detected in samples from least horseshoe bats. Samples from other bat species were negative for these viruses, indicating that the least horseshoe bat represents one of the natural reservoirs and mixers for strains of CoVs and has a pivotal role in the evolution and dissemination of these viruses. The genetic and evolutionary characteristics of these strains were described. Whole-genome sequencing of a new isolate (F46) from a rectal swab from a least horseshoe bat showed that it contained 29 699 nucleotides, excluding the poly (A) tail, with 13 open reading frames (ORFs). Phylogenetic and recombination analyses of F46 provided evidence of natural recombination between bat SARS-like CoVs (Rs3367 and LYRa11) or SARS-CoV (BJ01), suggesting that F46 could be a new recombinant virus from SARS-like CoVs or SARS-CoVs.

negative: Site-specific analysis of the SARS-CoV-2 glycan shield The emergence of the betacoronavirus, SARS-CoV-2 that causes COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, that mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in immune evasion and occluding immunogenic protein epitopes. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

negative: A Literature Review and Survey of Childhood Pneumonia Etiology Studies: 2000–2010 The Pneumonia Etiology Research for Child Health (PERCH) project is the largest multicountry etiology study of childhood pneumonia since the Board on Science and Technology in International Development studies of the 1980s. However, it is not the only recent or ongoing pneumonia etiology study, and even with seven sites, it cannot capture all epidemiologic settings in the developing world. Funding providers, researchers and policymakers rely on the best available evidence to strategically plan programs, new research directions and interventions. We aimed to describe the current landscape of recent pneumonia etiology studies in children under 5 years of age in the developed and developing world, as ascertained by a literature review of relevant studies with data since the year 2000 and a survey of researchers in the field of childhood pneumonia. We collected information on the study population, study design, case definitions, laboratory samples and methods and identified pathogens. A literature review identified 88 studies with child pneumonia etiology results. As of June 2010, our survey of researchers identified an additional 65 ongoing and recently completed child pneumonia etiology studies. This demonstrates the broad existing context into which the PERCH study must be placed. However, the landscape analysis also reveals a multiplicity of case definitions, levels of clinician involvement, facility types, specimen collection, and laboratory techniques. It reinforces the need for the standardization of methods and analyses for present and future pneumonia etiology studies in order to optimize their cumulative potential to accurately describe the microbial causes of childhood pneumonia.

negative: Interaction of Bordetella bronchiseptica and Its Lipopolysaccharide with In Vitro Culture of Respiratory Nasal Epithelium The nasal septa of fetal rabbits at 26 days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to Bordetella bronchiseptica or its lipopolysaccharide (LPS) for periods of 2 and 4 hours. A total of 240 explants were used. The tissues were examined using the Hematoxylin & Eosin technique. Then, semithin sections (0.5 μm) were stained with toluidine blue and examined with indirect immunoperoxidase (IPI) and lectin histochemistry. The most frequent and statistically significant findings were as follows: (1) cell death and increased goblet cell activity when exposed to bacteria and (2) cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to LPS. The lesions induced by the bacterium were more severe than with LPS alone, except for the cytoplasmic vacuolation in epithelial cells. IPI stained the ciliated border of the epithelium with the bacterium more intensely, while LPS lectin histochemistry preferentially labeled the cytoplasm of goblet cell. These data indicate that B. bronchiseptica and its LPS may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations.

negative: Potential Factors Influencing Repeated SARS Outbreaks in China Within last 17 years two widespread epidemics of severe acute respiratory syndrome (SARS) occurred in China, which were caused by related coronaviruses (CoVs): SARS-CoV and SARS-CoV-2. Although the origin(s) of these viruses are still unknown and their occurrences in nature are mysterious, some general patterns of their pathogenesis and epidemics are noticeable. Both viruses utilize the same receptor—angiotensin-converting enzyme 2 (ACE2)—for invading human bodies. Both epidemics occurred in cold dry winter seasons celebrated with major holidays, and started in regions where dietary consumption of wildlife is a fashion. Thus, if bats were the natural hosts of SARS-CoVs, cold temperature and low humidity in these times might provide conducive environmental conditions for prolonged viral survival in these regions concentrated with bats. The widespread existence of these bat-carried or -released viruses might have an easier time in breaking through human defenses when harsh winter makes human bodies more vulnerable. Once succeeding in making some initial human infections, spreading of the disease was made convenient with increased social gathering and holiday travel. These natural and social factors influenced the general progression and trajectory of the SARS epidemiology. However, some unique factors might also contribute to the origination of SARS in Wuhan. These factors are discussed in different scenarios in order to promote more research for achieving final validation.

negative: Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S(2) domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in S(D) domain) and G1124V (in S(2) subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird’s eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12038-020-00046-1) contains supplementary material, which is available to authorized users.

negative: Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR Background COVID-19 pandemic is underway. Some COVID-19 cases re-tested positive for SARS-CoV-2 RNA after discharge raising the public concern on their infectivity. Characterization of re-positive cases are urgently needed for designing intervention strategies. Methods Clinical data were obtained through Guangdong COVID-19 surveillance network. Neutralization antibody titre was determined using a microneutralization assay. Potential infectivity of clinical samples was evaluated after the cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. Results Among 619 discharged COVID-19 cases, 87 were re-tested as SARS-CoV-2 positive in circumstance of social isolation. All re-positive cases had mild or moderate symptoms in initial diagnosis and a younger age distribution (mean, 30.4). Re-positive cases (n=59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n=150) parallel-tested in this study. No infective viral strain could be obtained by culture and none full-length viral genomes could be sequenced for all re-positive cases. Conclusions Re-positive SARS-CoV-2 was not caused by the secondary infection and was identified in around 14% of discharged cases. A robust Nabs response and a potential virus genome degradation were detected from nearly all re-positive cases suggesting a lower transmission risk, especially through a respiratory route.

negative: The site of origin of the 1918 influenza pandemic and its public health implications

negative: Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. METHODS: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). RESULTS: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. CONCLUSIONS: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

negative: RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 'SARS-related-conserved' regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 'SARS-CoV-2-conserved-structured' regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5' UTR, frame-shifting element, and 3' UTR. Last, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 'SARS-CoV-2-conserved-unstructured' genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

negative: [The genome comparison of SARS-CoV and other coronaviruses]. The genome comparison of inter-species and intra-species can give us much information about the origin and evolution of viruses. There are 137 mutation sites in the 17 genomes of SARS-CoV,and the mutation rate is about 8.04 x 10(-3) substitution/site/year. The distribution of the segregating sites is not steady,the most variable region appears in S1 protein,and the nucleotide sequence of RNA-dependent RNA polymerase has very few mutation sites. The substitution bias of nucleotide acids and amino acids indicates the non-random drift products. The comparison of genome structures of SARS-CoV and other coronaviruses shows that SARS-CoV and IBV share the same genome structure. Phylogenetic analyses of conserved genes of coronaviruses indicate that SARS-CoV is a new branch of coronaviruses and appears more close to the group II coronaviruses. Interestingly,SARS-CoV shares some different features with different groups of coronaviruses. Additional analyses show that the first ORFs between S and E genes of some coronaviruses are transmembrane proteins and share the common motif,indicating the possible common ancestor. From the host distribution of different groups of coronaviruses and the phylogeny of s2m,we can deduce that avian is the probable natural host of SARS-CoV.

negative: Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV, and MERS-CoV genomes Abstract During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.

negative: Middle East respiratory syndrome coronavirus: transmission and phylogenetic evolution The Middle East respiratory syndrome coronavirus (MERS-CoV) was first described in 2012 and, subsequently, many cases were reported with a lower case fatality rate than initial cases. Humans can become infected within their communities and transmission can then be amplified in the healthcare setting. Contact investigation among cases shows a variable amount of spread among family members and healthcare workers. So far, circulating virus strains remain similar under continuous monitoring, with no genetic changes. Here, we discuss the transmission pattern, phylogenetic evolution, and pathogenesis of MERS-CoV infection.

negative: Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

negative: The Architecture of SARS-CoV-2 Transcriptome SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

negative: Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV studies have been conducted in China, more sequence information is needed to understand the molecular characterization of PDCoV. In this study, the partial ORF1a, spike protein (S) and nucleocapsid protein (N) were sequenced from Shandong Province between 2017 and 2018. The sequencing results for the S protein from 10 PDCoV strains showed 96.7 %-99.7 % nucleotide sequence identity with the China lineage strains, while sharing a lower level of nucleotide sequence identity, ranging from 95.7 to 96.8%, with the Vietnam/Laos/Thailand lineage strains. N protein sequencing analysis showed that these strains showed nucleotide homologies of 97.3%-99.3% with the reference strains. Phylogenetic analyses based on S protein sequences showed that these PDCoV strains were classified into the China lineage. The discontinuous 2 + 3 aa deletions at 400-401 and 758-760 were found in the Nsp2 and Nsp3 coding region in five strains, respectively, with similar deletions having been identified in Vietnam, Thailand, and Laos. Three novel patterns of deletion were observed for the first time in the Nsp2 and Nsp3 regions. Importantly, those findings suggest that PDCoV may have undergone a high degree of variation since PDCoV was first detected in China.

negative: Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010-2015 in Guangzhou Human coronavirus (HCoV) is one of the most common causes of respiratory tract infection throughout the world. To investigate the epidemiological and genetic variation of HCoV in Guangzhou, south China, we collected totally 13048 throat and nasal swab specimens from adults and children with fever and acute upper respiratory infection symptoms in Gunazhou, south China between July 2010 and June 2015, and the epidemiological features of HCoV and its species were studied. Specimens were screened for HCoV by real-time RT-PCR, and 7 other common respiratory viruses were tested simultaneously by PCR or real-time PCR. HCoV was detected in 294 cases (2.25%) of the 13048 samples, with most of them inpatients (251 cases, 85.4% of HCoV positive cases) and young children not in nursery (53.06%, 156 out of 294 HCoV positive cases). Four HCoVs, as OC43, 229E, NL63 and HKU1 were detected prevalent during 2010–2015 in Guangzhou, and among the HCoV positive cases, 60.20% were OC43, 16.67% were 229E, 14.97% were NL63 and 7.82% were HKU1. The month distribution showed that totally HCoV was prevalent in winter, but differences existed in different species. The 5 year distribution of HCoV showed a peak-valley distribution trend, with the detection rate higher in 2011 and 2013 whereas lower in 2010, 2012 and 2014. The age distribution revealed that children (especially those <3 years old) and old people (>50 years) were both high risk groups to be infected by HCoV. Of the 294 HCoV positive patients, 34.69% (101 cases) were co-infected by other common respiratory viruses, and influenza virus was the most common co-infecting virus (30/101, 29.70%). Fifteen HCoV-OC43 positive samples of 2013–2014 were selected for S gene sequencing and phylogenetic analysis, and the results showed that the 15 strains could be divided into 2 clusters in the phylogenetic tree, 12 strains of which formed a separate cluster that was closer to genotype G found in Malaysia. It was revealed for the first time that genotype B and genotype G of HCoV-OC43 co-circulated and the newly defined genotype G was epidemic as a dominant genotype during 2013–2014 in Guanzhou, south China.

negative: Treatment of COVID-19 Patients with Convalescent Plasma COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 – April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

negative: A molecular epidemiological study of human parainfluenza virus type 3 at a tertiary university hospital during 2013–2015 in Catalonia, Spain Abstract Human parainfluenza virus type 3 (HPIV-3) is one of the most common respiratory viruses particularly among young children and immunocompromised patients. The seasonality, prevalence and genetic diversity of HPIV-3 at a Spanish tertiary-hospital from 2013 to 2015 are reported. HPIV-3 infection was laboratory-confirmed in 102 patients (76%, under 5 years of age). Among <5 years-old patients, 9 (11.5%) were under any degree of immunosuppression, whereas this percentage was significantly higher (19; 79.2%) among patients older than 5 years. HPIV-3 was detected at varying levels, but mainly during spring and summer. All characterized HN/F sequences fell within C1b, C5 and in other two closely C3a-related groups. Furthermore, a new genetic lineage (C1c) was described. Genetic similarity and epidemiological data confirmed some nosocomial infections, highlighting the importance of the HPIV-3 surveillance, particularly in high-risk patients. This study provides valuable information on HPIV-3 diversity due to the scarce information in Europe.

negative: Polymorphism of SARS-CoV Genomes Abstract In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI Gen-Bank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3′ end (19.0 kb-29.7 kb). Regions encoding Orf10–11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27 700–28 000) which encodes parts of the putative ORF9 and ORF10–11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.

negative: SARS-CoV-2 orf1b Gene Sequence in the NTNG1 Gene on Human Chromosome 1 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.

negative: Bat origin of a new human coronavirus: there and back again

negative: Modeling site-specific amino-acid preferences deepens phylogenetic estimates of viral divergence Molecular phylogenetics is often used to estimate the time since the divergence of modern gene sequences. For highly diverged sequences, such phylogenetic techniques sometimes estimate surprisingly recent divergence times. In the case of viruses, independent evidence indicates that the estimates of deep divergence times from molecular phylogenetics are sometimes too recent. This discrepancy is caused in part by inadequate models of purifying selection leading to branch-length underestimation. Here we examine the effect on branch-length estimation of using models that incorporate experimental measurements of purifying selection. We find that models informed by experimentally measured site-specific amino-acid preferences estimate longer deep branches on phylogenies of influenza virus hemagglutinin. This lengthening of branches is due to more realistic stationary states of the models, and is mostly independent of the branch-length-extension from modeling site-to-site variation in amino-acid substitution rate. The branch-length extension from experimentally informed site-specific models is similar to that achieved by other approaches that allow the stationary state to vary across sites. However, the improvements from all of these site-specific but time-homogeneous and site-independent models are limited by the fact that a protein’s amino-acid preferences gradually shift as it evolves. Overall, our work underscores the importance of modeling site-specific amino-acid preferences when estimating deep divergence times—but also shows the inherent limitations of approaches that fail to account for how these preferences shift over time.

negative: Extensive diversity and evolution of hepadnaviruses in bats in China Abstract To better understand the evolution of hepadnaviruses, we sampled bats from Guizhou, Henan and Zhejiang provinces, China, and rodents from Zhejiang province. Genetically diverse hepadnaviruses were identified in a broad range of bat species, with an overall prevalence of 13.3%. In contrast, no rodent hepadnaviruses were identified. The newly discovered bat hepadnaviruses fell into two distinct phylogenetic groups. The viruses within the first group exhibited high diversity, with some closely related to viruses previously identified in Yunnan province. Strikingly, the newly discovered viruses sampled from Jiyuan city in the second phylogenetic group were most closely related to those found in bats from West Africa, suggestive of a long-term association between bats and hepadnaviruses. A co-phylogenetic analysis revealed frequent cross-species transmission among bats from different species, genera, and families. Overall, these data suggest that there are likely few barriers to the cross-species transmission of bat hepadnaviruses.

negative: A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Here we analyzed SARS-CoV-2 sequence diversity across 5,700 sequences sampled since December 2019. The Spike protein, which is the target immunogen of most vaccine candidates, showed 93 sites with shared polymorphisms; only one of these mutations was found in more than 1% of currently circulating sequences. The minimal diversity found among SARS-CoV-2 sequences can be explained by drift and bottleneck events as the virus spread away from its original epicenter in Wuhan, China. Importantly, there is little evidence that the virus has adapted to its human host since December 2019. Our findings suggest that a single vaccine should be efficacious against current global strains. One Sentence Summary The limited diversification of SARS-CoV-2 reflects drift and bottleneck events rather than adaptation to humans as the virus spread.

negative: Full genome characterization of two novel Alpha-coronavirus species from Italian bats Abstract Coronaviruses (CoVs) have been detected worldwide in several bat species, which are considered the main reservoir. The attention to the high diversity of CoVs hosted by bats has increased during the last decade due to the high number of human infections caused by two zoonotic Beta-CoVs, SARS-CoV and MERS-CoV, that cause several respiratory diseases. Among coronaviruses, two Alpha-CoV strains (HuCoV-229E and HuCoV-NL63) cause mild respiratory disease that can change to severe disease in children, elderly and individuals affected by illnesses. Phylogenetic analysis conducted on bat Alpha-CoV strains revealed their evolutive correlation to human strains, suggesting their origin in bats. The genome of CoVs is characterized by a high frequency of mutations and recombination events, increasing their ability to switch hosts and their zoonotic potential. In this study, three strains of Alpha-CoV genera detected in Italian bats (Pipistrellus kuhlii) were fully sequenced by Next Generation Sequencing (NGS) and characterized. The complete genome analysis showed the correlation of the Italians strains with a Chinese strain detected in 2013 and, based on CoV molecular species demarcation, two new Alpha-CoV species were established. The analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) showed the correlation of the Italian strains with CoVs that was only detected in the bat Pipistrellus genera (Pipistrellus kuhlii and Pipistrellus Pipistrellus) in European countries.

negative: SARS-CoV Infection Was from at Least Two Origins in the Taiwan Area OBJECTIVE: Severe acute respiratory syndrome (SARS) is caused by a new coronavirus. Genomic sequence analysis will provide the molecular epidemiology and help to develop vaccines. METHODS: We developed a rapid method to amplify and sequence the whole SARS-CoV genome from clinical specimens. The technique employed one-step multiplex RT-PCR to amplify the whole SARS-CoV genome, and then nested PCR was performed to amplify a 2-kb region separately. The PCR products were sequenced. RESULTS: We sequenced the genomes of SARS-CoV from 3 clinical specimens obtained in Taiwan. The sequences were similar to those reported by other groups, except that 17 single nucleotide variations and two 2-nucleotide deletions, and a 1-nucleotide deletion were found. All the variations in the clinical specimens did not alter the amino acid sequence. Of these 17 sequenced variants, two loci (positions 26203 and 27812) were segregated together as a specific genotype-T:T or C:C. Phylogenetic analysis showed two major clusters of SARS patients in Taiwan. CONCLUSION: We developed a very economical and rapid method to sequence the whole genome of SARS-CoV, which can avoid cultural influence. From our results, SARS patients in Taiwan may be infected from two different origins.

negative: The evolution of HIV: Inferences using phylogenetics Abstract Molecular phylogenetics has revolutionized the study of not only evolution but also disparate fields such as genomics, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry. Particularly significant are its achievements in population genetics as a result of the development of coalescent theory, which have contributed to more accurate model-based parameter estimation and explicit hypothesis testing. The study of the evolution of many microorganisms, and HIV in particular, have benefited from these new methodologies. HIV is well suited for such sophisticated population analyses because of its large population sizes, short generation times, high substitution rates and relatively small genomes. All these factors make HIV an ideal and fascinating model to study molecular evolution in real time. Here we review the significant advances made in HIV evolution through the application of phylogenetic approaches. We first examine the relative roles of mutation and recombination on the molecular evolution of HIV and its adaptive response to drug therapy and tissue allocation. We then review some of the fundamental questions in HIV evolution in relation to its origin and diversification and describe some of the insights gained using phylogenies. Finally, we show how phylogenetic analysis has advanced our knowledge of HIV dynamics (i.e., phylodynamics).

negative: Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.

negative: Characterization of a novel coronavirus associated with severe acute respiratory syndrome. In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.

negative: Polyphyletic origin of MERS coronaviruses and isolation of a novel clade A strain from dromedary camels in the United Arab Emirates Little is known regarding the molecular epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) circulating in dromedaries outside Saudi Arabia. To address this knowledge gap, we sequenced 10 complete genomes of MERS-CoVs isolated from 2 live and 8 dead dromedaries from different regions in the United Arab Emirates (UAE). Phylogenetic analysis revealed one novel clade A strain, the first detected in the UAE, and nine clade B strains. Strain D998/15 had a distinct phylogenetic position within clade A, being more closely related to the dromedary isolate NRCE-HKU205 from Egypt than to the human isolates EMC/2012 and Jordan-N3/2012. A comparison of predicted protein sequences also demonstrated the existence of two clade A lineages with unique amino acid substitutions, A1 (EMC/2012 and Jordan-N3/2012) and A2 (D998/15 and NRCE-HKU205), circulating in humans and camels, respectively. The nine clade B isolates belong to three distinct lineages: B1, B3 and B5. Two B3 strains, D1271/15 and D1189.1/15, showed evidence of recombination between lineages B4 and B5 in ORF1ab. Molecular clock analysis dated the time of the most recent common ancestor (tMRCA) of clade A to March 2011 and that of clade B to November 2011. Our data support a polyphyletic origin of MERS-CoV in dromedaries and the co-circulation of diverse MERS-CoVs including recombinant strains in the UAE.

negative: Coronavirus uses as binding site in humans angiotensin-converting enzyme 2 functional receptor that is involved in arterial blood pressure control and fibrotic response to damage and is a drug target in cardiovascular disease. Is this just a phylogenetic coincidence?

negative: Preliminary identification of potential vaccine targets for the COVID-19 coronavirus (SARS-CoV-2) based on SARS-CoV immunological studies The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the available SARS-CoV-2 sequences (as of 9 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.

negative: Median-joining network analysis of SARS-CoV-2 genomes is neither phylogenetic nor evolutionary

negative: A glimpse into the origins of genetic diversity in SARS-CoV-2

negative: A rapid, low cost, and highly sensitive SARS-CoV-2 diagnostic based on whole genome sequencing Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosis of SARS-CoV-2 infection, dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), that enhances sensitivity by aiming to amplify the entire SARS-CoV-2 genome rather than targeting particular viral loci, as in typical RT-PCR assays. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. We are able to reliably (>95% accuracy) detect SARS-CoV-2 at concentrations of 84 genome equivalents per milliliter, better than the reported limits of detection of almost all diagnostic methods currently approved by the US Food and Drug Administration. At higher concentrations, we are able to reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. Such genome assemblies enable the spread of the disease to be analyzed much more effectively than would be possible with an ordinary yes/no diagnostic, and can help identify vaccine and drug targets. Finally, we show that POLAR diagnoses on 10 of 10 clinical nasopharyngeal swab samples (half positive, half negative) match those obtained in a CLIA-certified lab using the Center for Disease Control’s 2019-Novel Coronavirus test. Using POLAR, a single person can process 192 samples over the course of an 8-hour experiment, at a cost of ∼$30/patient, enabling a 24-hour turnaround with sequencing and data analysis time included. Further testing and refinement will likely enable greater enhancements in the sensitivity of the above approach.

negative: CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/.

negative: Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

negative: Emergence of SARS-CoV-2 through Recombination and Strong Purifying Selection COVID-19 has become a global pandemic caused by a novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis and for drug discovery and vaccine development. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike gene and in other genes among bat, pangolin and human coronaviruses, indicating similar strong evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2’s entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2’s ability to infect humans. Similar purifying selection in different host species and frequent recombination among coronaviruses suggest a common evolutionary mechanism that could lead to new emerging human coronaviruses. One Sentence Summary Extensive Recombination and Strong Purifying Selection among coronaviruses from different hosts facilitate the emergence of SARS-CoV-2

negative: Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.

negative: Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak The Diamond Princess (DP) cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a COVID-19 case. We performed whole genome sequencing of SARS-CoV-2 directly from PCR-positive clinical specimens and conducted a haplotype network analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the DP originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters were linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network analysis in identifying potential infection routes.

negative: Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China Abstract Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV studies have been conducted in China, more sequence information is needed to understand the molecular characterization of PDCoV. In this study, the partial ORF1a, spike protein (S) and nucleocapsid protein (N) were sequenced from Shandong Province between 2017 and 2018. The sequencing results for the S protein from 10 PDCoV strains showed 96.7 %–99.7 % nucleotide sequence identity with the China lineage strains, while sharing a lower level of nucleotide sequence identity, ranging from 95.7 to 96.8%, with the Vietnam/Laos/Thailand lineage strains. N protein sequencing analysis showed that these strains showed nucleotide homologies of 97.3%–99.3% with the reference strains. Phylogenetic analyses based on S protein sequences showed that these PDCoV strains were classified into the China lineage. The discontinuous 2 + 3 aa deletions at 400–401 and 758–760 were found in the Nsp2 and Nsp3 coding region in five strains, respectively, with similar deletions having been identified in Vietnam, Thailand, and Laos. Three novel patterns of deletion were observed for the first time in the Nsp2 and Nsp3 regions. Importantly, those findings suggest that PDCoV may have undergone a high degree of variation since PDCoV was first detected in China.

negative: Comparative analysis of the SARS coronavirus genome: a good start to a long journey

negative: Phylogenetic and epidemic modeling of rapidly evolving infectious diseases Abstract Epidemic modeling of infectious diseases has a long history in both theoretical and empirical research. However the recent explosion of genetic data has revealed the rapid rate of evolution that many populations of infectious agents undergo and has underscored the need to consider both evolutionary and ecological processes on the same time scale. Mathematical epidemiology has applied dynamical models to study infectious epidemics, but these models have tended not to exploit – or take into account – evolutionary changes and their effect on the ecological processes and population dynamics of the infectious agent. On the other hand, statistical phylogenetics has increasingly been applied to the study of infectious agents. This approach is based on phylogenetics, molecular clocks, genealogy-based population genetics and phylogeography. Bayesian Markov chain Monte Carlo and related computational tools have been the primary source of advances in these statistical phylogenetic approaches. Recently the first tentative steps have been taken to reconcile these two theoretical approaches. We survey the Bayesian phylogenetic approach to epidemic modeling of infection diseases and describe the contrasts it provides to mathematical epidemiology as well as emphasize the significance of the future unification of these two fields.

negative: Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2 The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.

negative: A Patient with Asymptomatic Severe Acute Respiratory Syndrome (SARS) and Antigenemia from the 2003–2004 Community Outbreak of SARS in Guangzhou, China An asymptomatic case of severe acute respiratory syndrome (SARS) occurred early in 2004, during a community outbreak of SARS in Guangzhou, China. This was the first time that a case of asymptomatic SARS was noted in an individual with antigenemia and seroconversion. The asymptomatic case patient and the second index case patient with SARS in the 2003–2004 outbreak both worked in the same restaurant, where they served palm civets, which were found to carry SARS-associated coronaviruses. Epidemiological information and laboratory findings suggested that the findings for the patient with asymptomatic infection, together with the findings from previously reported serological analyses of handlers of wild animals and the 4 index case patients from the 2004 community outbreak, reflected a likely intermediate phase of animal-to-human transmission of infection, rather than a case of human-to-human transmission. This intermediate phase may be a critical stage for virus evolution and disease prevention.

negative: Genomic diversity of SARS-CoV-2 in Coronavirus Disease 2019 patients BACKGROUND: A novel coronavirus (SARS-CoV-2) has infected more than 75,000 individuals and spread to over 20 countries. It is still unclear how fast the virus evolved and how the virus interacts with other microorganisms in the lung. METHODS: We have conducted metatranscriptome sequencing for the bronchoalveolar lavage fluid of eight SARS-CoV-2 patients, 25 community-acquired pneumonia (CAP) patients, and 20 healthy controls. RESULTS: The median number of intra-host variants was 1-4 in SARS-CoV-2 infected patients, which ranged between 0 and 51 in different samples. The distribution of variants on genes was similar to those observed in the population data (110 sequences). However, very few intra-host variants were observed in the population as polymorphism, implying either a bottleneck or purifying selection involved in the transmission of the virus, or a consequence of the limited diversity represented in the current polymorphism data. Although current evidence did not support the transmission of intra-host variants in a person-to-person spread, the risk should not be overlooked. The microbiota in SARS-CoV-2 infected patients was similar to those in CAP, either dominated by the pathogens or with elevated levels of oral and upper respiratory commensal bacteria. CONCLUSION: SARS-CoV-2 evolves in vivo after infection, which may affect its virulence, infectivity, and transmissibility. Although how the intra-host variant spreads in the population is still elusive, it is necessary to strengthen the surveillance of the viral evolution in the population and associated clinical changes.

negative: Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes SUMMARY: Genome detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next-generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present the Genome Detective Coronavirus Typing Tool that can accurately identify the novel severe acute respiratory syndrome (SARS)-related coronavirus (SARS-CoV-2) sequences isolated in China and around the world. The tool can accept up to 2000 sequences per submission and the analysis of a new whole-genome sequence will take approximately 1 min. The tool has been tested and validated with hundreds of whole genomes from 10 coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for SARS-CoV-2. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines to stop the COVID-19 disease. AVAILABILITY AND IMPLEMENTATION: https://www.genomedetective.com/app/typingtool/cov. CONTACT: koen@emweb.be or deoliveira@ukzn.ac.za. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

negative: A tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses World Health Organization has declared the ongoing outbreak of coronavirus disease 2019 (COVID-19) a Public Health Emergency of International Concern. The virus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. Human infection with SARS-CoV-2 leads to a wide range of clinical manifestations ranging from asymptomatic, mild, moderate to severe. The severe cases present with pneumonia, which can progress to acute respiratory distress syndrome. The outbreak provides an opportunity for real-time tracking of an animal coronavirus that has just crossed species barrier to infect humans. The outcome of SARS-CoV-2 infection is largely determined by virus-host interaction. Here, we review the discovery, zoonotic origin, animal hosts, transmissibility and pathogenicity of SARS-CoV-2 in relation to its interplay with host antiviral defense. A comparison with SARS-CoV, Middle East respiratory syndrome coronavirus, community-acquired human coronaviruses and other pathogenic viruses including human immunodeficiency viruses is made. We summarize current understanding of the induction of a proinflammatory cytokine storm by other highly pathogenic human coronaviruses, their adaptation to humans and their usurpation of the cell death programmes. Important questions concerning the interaction between SARS-CoV-2 and host antiviral defence, including asymptomatic and presymptomatic virus shedding, are also discussed.

negative: Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease Abstract The severe acute respiratory syndrome coronavirus (SARS-CoV) caused substantial morbidity and mortality in 2002–2003. Deletion of the envelope (E) protein modestly diminished virus growth in tissue culture but abrogated virulence in animals. Here, we show that immunization with rSARS-CoV-ΔE or SARS-CoV-Δ[E,6-9b] (deleted in accessory proteins (6, 7a, 7b, 8a, 8b, 9b) in addition to E) nearly completely protected BALB/c mice from fatal respiratory disease caused by mouse-adapted SARS-CoV and partly protected hACE2 Tg mice from lethal disease. hACE2 Tg mice, which express the human SARS-CoV receptor, are extremely susceptible to infection. We also show that rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] induced anti-virus T cell and antibody responses. Further, the E-deleted viruses were stable after 16 blind passages through tissue culture cells, with only a single mutation in the surface glycoprotein detected. The passaged virus remained avirulent in mice. These results suggest that rSARS-CoV-ΔE is an efficacious vaccine candidate that might be useful if SARS recurred.

negative: Molecular Evolution of Human Coronavirus Genomes Human coronaviruses (HCoVs), including SARS-CoV and MERS-CoV, are zoonotic pathogens that originated in wild animals. HCoVs have large genomes that encode a fixed array of structural and nonstructural components, as well as a variety of accessory proteins that differ in number and sequence even among closely related CoVs. Thus, in addition to recombination and mutation, HCoV genomes evolve through gene gains and losses. In this review we summarize recent findings on the molecular evolution of HCoV genomes, with special attention to recombination and adaptive events that generated new viral species and contributed to host shifts and to HCoV emergence. Video Abstract

negative: In silico identification of conserved cis-acting RNA elements in the SARS-CoV-2 genome Aim The aim of this study was to computationally predict conserved RNA sequences and structures known as cis-acting RNA elements (CREs) located within the SARS-CoV-2 genome. Materials & methods Bioinformatics tools were used to analyse and predict cis-acting regulatory elements by obtaining viral sequences from available databases. Results Computational analysis prediction revealed the presence of RNA stem-loop structures within the 3’ end of the ORF1ab region that are analogous to the previously identified SARS-CoV genomic packaging signals. Alignment-based RNA secondary structures prediction of the 5’ end of the SARS-CoV-2 genome identified also conserved CREs. Conclusion These CREs could be used as potential targets for a vaccine and/or antiviral therapeutics developments; however, further studies would be required to confirm their roles in the SARS-CoV-2 life cycle.

negative: DV-Curve Representation of Protein Sequences and Its Application Based on the detailed hydrophobic-hydrophilic(HP) model of amino acids, we propose dual-vector curve (DV-curve) representation of protein sequences, which uses two vectors to represent one alphabet of protein sequences. This graphical representation not only avoids degeneracy, but also has good visualization no matter how long these sequences are, and can reflect the length of protein sequence. Then we transform the 2D-graphical representation into a numerical characterization that can facilitate quantitative comparison of protein sequences. The utility of this approach is illustrated by two examples: one is similarity/dissimilarity comparison among different ND6 protein sequences based on their DV-curve figures the other is the phylogenetic analysis among coronaviruses based on their spike proteins.

negative: SARS-CoV-2 Whole Genome Amplification and Sequencing for Effective Population-Based Surveillance and Control of Viral Transmission Background With the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing COVID-19 pandemic. Unlike RT-qPCR, SARS-CoV-2 Whole Genome Sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. However, practical and cost considerations of cWGS should be addressed before it can be widely implemented. Methods We performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 full genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. To track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled SARS-CoV-2 genomes to publicly available sequences. Results We show a significant improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of SARS-CoV-2. With enrichment, more than 99% of the sequencing reads mapped to the viral genome compared to an average of 0.63% without enrichment. Consequently, a dramatic increase in genome coverage was obtained using significantly less sequencing data, enabling higher scalability and significant cost reductions. We also demonstrate how SARS-CoV-2 genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains. Conclusions SARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic.

negative: Multiomics analysis of the giant triton snail salivary gland, a crown-of-thorns starfish predator The giant triton snail (Charonia tritonis) is one of the few natural predators of the adult Crown-of-Thorns starfish (COTS), a corallivore that has been damaging to many reefs in the Indo-Pacific. Charonia species have large salivary glands (SGs) that are suspected to produce either a venom and/or sulphuric acid which can immobilize their prey and neutralize the intrinsic toxic properties of COTS. To date, there is little information on the types of toxins produced by tritons. In this paper, the predatory behaviour of the C. tritonis is described. Then, the C. tritonis SG, which itself is made up of an anterior lobe (AL) and posterior lobe (PL), was analyzed using an integrated transcriptomics and proteomics approach, to identify putative toxin- and feeding-related proteins. A de novo transcriptome database and in silico protein analysis predicts that ~3800 proteins have features consistent with being secreted. A gland-specific proteomics analysis confirmed the presence of numerous SG-AL and SG-PL proteins, including those with similarity to cysteine-rich venom proteins. Sulfuric acid biosynthesis enzymes were identified, specific to the SG-PL. Our analysis of the C. tritonis SG (AL and PL) has provided a deeper insight into the biomolecular toolkit used for predation and feeding by C. tritonis.

negative: Genetic analysis of SARS-CoV-2 isolates collected from Bangladesh: insights into the origin, mutation spectrum, and possible pathomechanism As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of 14 isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and the Middle East, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant interaction was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.

negative: Evolution and Variation of the SARS-CoV Genome Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

negative: [Variability analysis of S2 gene of SARS-CoV]. OBJECTIVE To determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene. METHOD S2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database. RESULT With the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope. CONCLUSION S2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.

negative: Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson’s leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs.

negative: Complete Genome Sequences of SARS-CoV-2 Strains Detected in Malaysia We sequenced four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Malaysia during the second wave of infection and found unique mutations which suggest local evolution. Circulating Malaysian strains represent introductions from different countries, particularly during the first wave of infection. Genome sequencing is important for understanding local epidemiology.

negative: Achievements and highlights for Infection, Disease and Health

negative: In the frame, road map for Australian sport on an uncertain journey through COVID-19

negative: Reply to “Comments on Fouchier’s Calculation of Risk and Elapsed Time for Escape of a Laboratory-Acquired Infection from His Laboratory”

negative: Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites Abstract The outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus continually led to worldwide human infections and deaths. Currently, there is no specific viral protein-targeted therapeutics. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein remains unclear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

negative: The role of masks and respirator protection against SARS-CoV-2

negative: Host range of SARS-CoV-2 and implications for public health

negative: Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated Coronavirus disease 2019 (COVID-19) pandemic has demanded rapid upscaling of in-vitro diagnostic assays to enable mass screening and testing of high-risk groups, and simultaneous ascertainment of robust data on past SARS-CoV-2 exposure at an individual and population level. To meet the exponential demand in testing, there has been an accelerated development of both molecular and serological assays across a plethora of platforms. In the present review, we discuss the current literature on these modalities including the nucleic acid amplification tests, direct viral antigen tests and the rapidly expanding laboratory based and point of care serological tests. This suite of complementary tests will inform crucial decisions by healthcare providers and policy makers and understanding their strengths and limitations will be critical to their judicious application for the development of algorithmic approaches to treatment and public health strategies.

negative: The relationship of severe acute respiratory syndrome coronavirus with avian and other coronaviruses. In February 2003, a severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in humans in Guangdong Province, China, and caused an epidemic that had severe impact on public health, travel, and economic trade. Coronaviruses are worldwide in distribution, highly infectious, and extremely difficult to control because they have extensive genetic diversity, a short generation time, and a high mutation rate. They can cause respiratory, enteric, and in some cases hepatic and neurological diseases in a wide variety of animals and humans. An enormous, previously unrecognized reservoir of coronaviruses exists among animals. Because coronaviruses have been shown, both experimentally and in nature, to undergo genetic mutations and recombination at a rate similar to that of influenza viruses, it is not surprising that zoonosis and host switching that leads to epidemic diseases have occurred among coronaviruses. Analysis of coronavirus genomic sequence data indicates that SARS-CoV emerged from an animal reservoir. Scientists examining coronavirus isolates from a variety of animals in and around Guangdong Province reported that SARS-CoV has similarities with many different coronaviruses including avian coronaviruses and SARS-CoV-like viruses from a variety of mammals found in live-animal markets. Although a SARS-like coronavirus isolated from a bat is thought to be the progenitor of SARS-CoV, a lack of genomic sequences for the animal coronaviruses has prevented elucidation of the true origin of SARS-CoV. Sequence analysis of SARS-CoV shows that the 5' polymerase gene has a mammalian ancestry; whereas the 3' end structural genes (excluding the spike glycoprotein) have an avian origin. Spike glycoprotein, the host cell attachment viral surface protein, was shown to be a mosaic of feline coronavirus and avian coronavirus sequences resulting from a recombination event. Based on phylogenetic analysis designed to elucidate evolutionary links among viruses, SARS-CoV is believed to have branched from the modern Group 2 coronaviruses, suggesting that it evolved relatively rapidly. This is significant because SARS-CoV is likely still circulating in an animal reservoir (or reservoirs) and has the potential to quickly emerge and cause a new epidemic.

negative: The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 The 21st International Symposium on Intensive Care and Emergency Medicine was dominated by the results of recent clinical trials in sepsis and acute respiratory distress syndrome (ARDS). The promise of extracorporeal liver replacement therapy and noninvasive ventilation were other areas of interest. Ethical issues also received attention. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of a high standard. The meeting was marked by a sense of renewed enthusiasm that positive progress is occurring in intensive care medicine.

negative: Date of origin of the SARS coronavirus strains BACKGROUND: A new respiratory infectious epidemic, severe acute respiratory syndrome (SARS), broke out and spread throughout the world. By now the putative pathogen of SARS has been identified as a new coronavirus, a single positive-strand RNA virus. RNA viruses commonly have a high rate of genetic mutation. It is therefore important to know the mutation rate of the SARS coronavirus as it spreads through the population. Moreover, finding a date for the last common ancestor of SARS coronavirus strains would be useful for understanding the circumstances surrounding the emergence of the SARS pandemic and the rate at which SARS coronavirus diverge. METHODS: We propose a mathematical model to estimate the evolution rate of the SARS coronavirus genome and the time of the last common ancestor of the sequenced SARS strains. Under some common assumptions and justifiable simplifications, a few simple equations incorporating the evolution rate (K) and time of the last common ancestor of the strains (T(0)) can be deduced. We then implemented the least square method to estimate K and T(0 )from the dataset of sequences and corresponding times. Monte Carlo stimulation was employed to discuss the results. RESULTS: Based on 6 strains with accurate dates of host death, we estimated the time of the last common ancestor to be about August or September 2002, and the evolution rate to be about 0.16 base/day, that is, the SARS coronavirus would on average change a base every seven days. We validated our method by dividing the strains into two groups, which coincided with the results from comparative genomics. CONCLUSION: The applied method is simple to implement and avoid the difficulty and subjectivity of choosing the root of phylogenetic tree. Based on 6 strains with accurate date of host death, we estimated a time of the last common ancestor, which is coincident with epidemic investigations, and an evolution rate in the same range as that reported for the HIV-1 virus.

negative: Nipah shell disorder, modes of infection, and virulence Abstract The Nipah Virus (NiV) was first isolated during a 1998–9 outbreak in Malaysia. The outbreak initially infected farm pigs and then moved to humans from pigs with a case-fatality rate (CFR) of about 40%. After 2001, regular outbreaks occurred with higher CFRs (~71%, 2001–5, ~93%, 2008–12). The spread arose from drinking virus-laden palm date sap and human-to-human transmission. Intrinsic disorder analysis revealed strong correlation between the percentage of disorder in the N protein and CFR (Regression: r2 = 0.93, p < 0.01, ANOVA: p < 0.01). Distinct disorder and, therefore, genetic differences can be found in all three group of strains. The fact that the transmission modes of the Malaysia strain are different from those of the Bangladesh strains suggests that the correlations may also be linked to the modes of viral transmission. Analysis of the NiV and related viruses suggests links between modes of transmission and disorder of not just the N protein but, also, of M shell protein. The links among shell disorder, transmission modes, and virulence suggest mechanisms by which viruses are attenuated as they passed through different cell hosts from different animal species. These have implications for development of vaccines and epidemiological molecular analytical tools to contain outbreaks.

negative: SARS-CoV-2 orthologs of pathogenesis-involved small viral RNAs of SARS-CoV Background: The COVID-19 pandemic clock is ticking and the survival of many of mankind's modern institutions and or survival of many individuals is at stake. There is a need for treatments to significantly reduce the morbidity and mortality of COVID-19. Hence, we delved deep into the SARS-CoV-2 genome, which is the virus that has caused COVID-19. SARS-CoV-2 is from the same family as SARS-CoV in which three small viral RNAs (svRNA) were recently identified; those svRNAs play a significant role in the virus pathogenesis in mice. Contribution: In this paper, we report potential orthologs of those three svRNAs in the SARS-CoV-2 genome. Instead of off-the-shelf search and alignment algorithms, which failed to discover the orthologs, we used a special alignment scoring that does not penalize C/T and A/G mismatches. RNA bases C and U both can bind to G; similarly, A and G both can bind to U, hence, our scoring. To validate our results, we confirmed the discovered orthologs are fully conserved in all the publicly available genomes of various strains of SARS-CoV-2; the loci at which the SARS-CoV-2 orthologs occur are close to the loci at which SARS-CoV svRNAs occur. We also report potential targets for these svRNAs. We hypothesize that the discovered orthologs play a role in pathogenesis of SARS-CoV-2, and therefore, antagomir-mediated inhibition of these SARS-CoV-2 svRNAs inhibits COVID-19.

negative: Mapping population mental health concerns related to COVID-19 and the consequences of physical distancing: a Google trends analysis Background: The 2020 Coronavirus pandemic is a major international public health challenge. Governments have taken public health protection measures to reduce the spread of the virus through non-pharmalogical measures. The impact of the pandemic and the public health response on individual and population mental health is unknown. Methods: We used Google Trends data (1 Jan 2020 - 30 Mar 2020) to investigate the impact of the pandemic and government measures to curb it on people’s concerns, as indexed by changes in search frequency for topics indicating mental distress, social and economic stressors and mental health treatment-seeking. We explored the changes of key topics in Google trends in Italy, Spain, USA, UK, and Worldwide in relation to sentinel events during the pandemic. Results: Globally there appears to be significant concerns over the financial and work-related consequences of the pandemic, with some evidence that levels of fear are rising. Conversely relative searching for topics related to depression and suicide fell after the pandemic was announced, with some evidence that searches for the latter have risen recently. Concerns over education and access to medication appear to be particular social stressors. Whilst searches for face-to-face treatments have declined, those for self-care have risen. Conclusions: Monitoring Google trends shows promise as a means of tracking changing public concerns. In weeks to come it may enable policy makers to assess the impact of their interventions including those aiming to limit negative consequences, such as government funded financial safety nets.

negative: Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection Abstract SARS-CoV-2 causes the recent global COVID-19 public health emergency. ACE2 is the receptor for both SARS-CoV-2 and SARS-CoV. To predict the potential host range of SARS-CoV-2, we analyzed the key residues of ACE2 for recognizing S protein. We found that most of the selected mammals including pets (dog and cat), pangolin and Circetidae mammals remained the most of key residues for association with S protein from SARS-CoV and SARS-CoV-2. The interaction interface between cat/dog/pangolin/Chinese hamster ACE2 and SARS-CoV/SARS-CoV-2 S protein was simulated through homology modeling. We identified that N82 in ACE2 showed a closer contact with SARS-CoV-2 S protein than M82 in human ACE2. Our finding will provide important insights into the host range of SARS-CoV-2 and a new strategy to design an optimized ACE2 for SARS-CoV-2 infection.

negative: Development of a risk assessment tool for contact tracing people after contact with infectious patients while travelling by bus or other public ground transport: a Delphi consensus approach BACKGROUND: Tracing persons who have been in contact with an infectious patient may be very effective in preventing the spread of communicable diseases. However, criteria to decide when to conduct contact tracing are not well established. We have investigated the available evidence for contact tracing with a focus on public ground transport aiming to give guidance in what situations contact tracing should be considered. METHODS: Relevant infectious diseases suitable for contact tracing in ground transport and a set of disease-specific epidemiological criteria were defined through literature search and structured multistep expert consultations. We developed continuous scales for each criterion to be rated for its relevance to contact tracing in ground transport. We used the Delphi method with an international expert panel to position the values of criteria on the respective scales. RESULTS: The study led to the development of the ‘Contact Tracing-Risk Assessment Profile’ (CT-RAP), a decision-making instrument, taking into account pathogen-specific as well as situation-specific criteria. This report describes the methodology of this instrument and presents two examples of ready-to-use CT-RAP for tuberculosis and for meningococcal disease in public ground transport. DISCUSSION: The systematic and transparent use of the CT-RAP for tuberculosis and meningococcal disease is likely to facilitate reasonable, efficient and user-friendly decisions with respect to contact tracing. New CT-RAPs for additional pathogens and different settings such as schools and kindergartens are being planned.

negative: The mechanistic overview of SARS-CoV-2 using angiotensin-converting enzyme 2 to enter the cell for replication: possible treatment options related to the renin–angiotensin system The SARS-CoV-2 pandemic is a healthcare crisis caused by insufficient knowledge applicable to effectively combat the virus. Therefore, different scientific discovery strategies need to be connected, to generate a rational treatment which can be made available as rapidly as possible. This relies on a solid theoretical understanding of the mechanisms of SARS-CoV-2 infection and host responses, which is coupled to the practical experience of clinicians that are treating patients. Because SARS-CoV-2 enters the cell by binding to angiotensin-converting enzyme 2 (ACE2), targeting ACE2 to prevent such binding seems an obvious strategy to combat infection. However, ACE2 performs its functions outside the cell and was found to enter the cell only by angiotensin II type 1 receptor (AT1R)-induced endocytosis, after which ACE2 is destroyed. This means that preventing uptake of ACE2 into the cell by blocking AT1R would be a more logical approach to limit entry of SARS-CoV-2 into the cell. Since ACE2 plays an important protective role in maintaining key biological processes, treatments should not disrupt the functional capacity of ACE2, to counterbalance the negative effects of the infection. Based on known mechanisms and knowledge of the characteristics of SARS-CoV we propose the hypothesis that the immune system facilitates SARS-CoV-2 replication which disrupts immune regulatory mechanisms. The proposed mechanism by which SARS-CoV-2 causes disease immediately suggests a possible treatment, since the AT1R is a key player in this whole process. AT1R antagonists appear to be the ideal candidate for the treatment of SARS-CoV-2 infection. AT1R antagonists counterbalance the negative consequences of angiotesnin II and, in addition, they might even be involved in preventing the cellular uptake of the virus without interfering with ACE2 function. AT1R antagonists are widely available, cheap, and safe. Therefore, we propose to consider using AT1R antagonists in the treatment of SARS-CoV-2.

negative: Outbreak of SARS-CoV2: Pathogenesis of infection and cardiovascular involvement Since the new coronavirus SARS (SARS-CoV2) has emerged from china, the infection (COVID-19) has affected many countries and led to many deaths worldwide. Like SARS-CoV, ACE2 as a functional receptor for SARS-CoV2 is essential for virus to entry to the cell. ACE2 is a part of RAS which is expressed in several organs that opposes the AngII functions by converting Ang II to Ang (1-7), the one with vasodilation effects. The death rate of COVID-19 is estimated about 3.4%, however, some comorbid conditions like underlying cardiovascular disease, hypertension, and diabetes increase the risk of mortality. In addition, cardiovascular involvement by SARS-CoV2 could be direct through either ACE2 receptors which are expressed tremendously in heart, or by the surge of different cytokines or by ARDS-induced hypoxia. Traditional risk factors could aggravate the process of COVID-19 infection that urges the triage of these high risk patients for SARS-CoV2. Currently, there is no effective, proven treatment or vaccination for COVID-19, but many investigators are struggling to find a treatment strategy as soon as possible. Some potential medications like chloroquine by itself or in combination with azithromycin, and some protease inhibitors used for the treatment of COVID-19 have cardiovascular adverse effects that should be kept in mind in order to close monitor of the patients receiving these medications.

negative: Synthesis and evaluation of isatin derivatives as effective SARS coronavirus 3CL protease inhibitors Abstract N-Substituted isatin derivatives were prepared from the reaction of isatin and various bromides via two steps. Bioactivity assay results (in vitro tests) demonstrated that some of these compounds are potent and selective inhibitors against SARS coronavirus 3CL protease with IC50 values ranging from 0.95 to 17.50μM. Additionally, isatin 4o exhibited more potent inhibition for SARS coronavirus protease than for other proteases including papain, chymotrypsin, and trypsin.

negative: Inhibitors of SARS-CoV-2 Entry: Current and Future Opportunities [Image: see text] Recently, a novel coronavirus initially designated 2019-nCoV but now termed SARS-CoV-2 has emerged and raised global concerns due to its virulence. SARS-CoV-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to COVID-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the World Health Organization (WHO). At this time, no specific prophylactic or postexposure therapy for COVID-19 are currently available. Viral entry is the first step in the SARS-CoV-2 lifecycle and is mediated by the trimeric spike protein. Being the first stage in infection, entry of SARS-CoV-2 into host cells is an extremely attractive therapeutic intervention point. Within this review, we highlight therapeutic intervention strategies for anti-SARS-CoV, MERS-CoV, and other coronaviruses and speculate upon future directions for SARS-CoV-2 entry inhibitor designs.

negative: Molecular epidemiology of infectious bronchitis virus in Poland from 1980 to 2017 Abstract The presence of infectious bronchitis virus (IBV) was identified for the first time in the poultry population in Poland at the end of the 1960s. From this time a few waves of epidemics caused by different IBV variants spread across the country. In order to gain more insight into the molecular epidemiology of IBV in Poland, in the present study the S1 coding region of 34 IBV isolates and nearly whole genome of 10 strains collected over a period of 38 years was characterized. Phylogenetic analysis showed that these strains belonged to five recently established IBV lineages: GI-1, GI-12, GI-13, GI-19 and GI-23. Additionally, two strains from 1989 and 1997 formed a separate branch of the phylogenetic tree categorized as unique early Polish variants, and one strain was revealed to be the recombinant of these and GI-1 lineage viruses. Irrespective of year of isolation and S1-dependent genotype, the genome sequences of Polish IBV strains showed the presence of six genes and 13 ORFs: 5′UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR, however their individual genes and putative proteins had different lengths. The phylogenetic analyses performed on the genome of ten Polish IBV strains revealed that they cluster into different groups. The Polish GI-1, GI-19 and GI-23 strains cluster with other similar viruses of these lineages, with the exception of the two strains from 1989 and 1997 which are different. It seems that in Poland in the 1980s and 1990s IBV strains with a unique genome backbone circulated in the field, which were then replaced by other strains belonging to other IBV lineages with a genome backbone specific to these lineages. The recombination analysis showed that some Polish strains resulted from a recombination event involving different IBV lineages, most frequently GI-13 and GI-19.

negative: Genetic diversity and phylogenetic analysis of newly discovered bat astroviruses in Korea Bats have been identified as a natural reservoir for several potentially zoonotic viruses. Recently, astroviruses have been reported in bats in many countries, but not Korea. We collected 363 bat samples from thirteen species at twenty-nine sites in Korea across 2016 and tested them for astrovirus. The detection of the RNA-dependent RNA polymerase (RdRp) gene in bat astroviruses was confirmed in thirty-four bats across four bat species in Korea: twenty-five from Miniopterus fuliginosusi, one from Myotis macrodactylus, four from M. petax, and four from Rhinolophus ferrumequinum. The highest detection rates for astrovirus were found in Sunchang (61.5%, 8/13 bats), and in the samples collected in April (63.2%, 12/19 bats). The amino acid identity of astroviral sequences identified from bat samples was ≥ 46.6%. More specifically, the amino acid identity within multiple clones from individual bats was ≥ 50.8%. Additionally, the phylogenetic topology between astroviruses from different bat families showed a close relationship. Furthermore, phylogenetic analysis of the partial ORF2 sequence of bat astroviruses was found to have a maximum similarity of 73.3–74.8% with available bat astrovirus sequences. These results indicate potential multiple-infection by several bat astrovirus species in individual bats, or hyperpolymorphism in the astrovirus strains, as well as the transmission of astroviruses across bat families; furthermore, our phylogenetic analysis of the partial ORF2 implied that a novel astrovirus may exist. However, the wide diversity of astroviral sequences appeared to have no significant correlation with bat species or the spatiotemporal distribution of Korean bat astroviruses.

negative: Evolutionary characterization of the emerging porcine epidemic diarrhea virus worldwide and 2014 epidemic in Taiwan Abstract Since 2010, a new variant of PEDV belonging to Genogroup 2 has been transmitting in China and further spreading to the Unites States and other Asian countries including Taiwan. In order to characterize in detail the temporal and geographic relationships among PEDV strains, the present study systematically evaluated the evolutionary patterns and phylogenetic resolution in each gene of the whole PEDV genome in order to determine which regions provided the maximal interpretative power. The result was further applied to identify the origin of PEDV that caused the 2014 epidemic in Taiwan. Thirty-four full genome sequences were downloaded from GenBank and divided into three non-mutually exclusive groups, namely, worldwide, Genogroup 2 and China, to cover different ranges of secular and spatial trends. Each dataset was then divided into different alignments by different genes for likelihood mapping and phylogenetic analysis. Our study suggested that both nsp3 and S genes contained the highest phylogenetic signal with substitution rate and phylogenetic topology similar to those obtained from the complete genome. Furthermore, the proportion of nodes with high posterior support (posterior probability >0.8) was similar between nsp3 and S genes. The nsp3 gene sequences from three clinical samples of swine with PEDV infections were aligned with other strains available from GenBank and the results suggested that the virus responsible for the 2014 PEDV outbreak in Taiwan clustered together with Clade I from the US within Genogroup 2. In conclusion, the current study identified the nsp3 gene as an alternative marker for a rapid and unequivocal classification of the circulating PEDV strains which provides complementary information to the S gene in identifying the emergence of epidemic strain resulting from recombination.

negative: Distant Relatives of Severe Acute Respiratory Syndrome Coronavirus and Close Relatives of Human Coronavirus 229E in Bats, Ghana We tested 12 bat species in Ghana for coronavirus (CoV) RNA. The virus prevalence in insectivorous bats (n = 123) was 9.76%. CoV was not detected in 212 fecal samples from Eidolon helvum fruit bats. Leaf-nosed bats pertaining to Hipposideros ruber by morphology had group 1 and group 2 CoVs. Virus concentrations were <45,000 copies/100 mg of bat feces. The diversified group 1 CoV shared a common ancestor with the human common cold virus hCoV-229E but not with hCoV-NL63, disputing hypotheses of common human descent. The most recent common ancestor of hCoV-229E and GhanaBt-CoVGrp1 existed in ≈1686–1800 ad. The GhanaBt-CoVGrp2 shared an old ancestor (≈2,400 years) with the severe acute respiratory syndrome–like group of CoV.

negative: Genetic Grouping of SARS-CoV-2 Coronavirus Sequences using Informative Subtype Markers for Pandemic Spread Visualization We propose an efficient framework for genetic subtyping of SARS-CoV-2, the novel coronavirus that causes the COVID-19 pandemic. Efficient viral subtyping enables visualization and modeling of the geographic distribution and temporal dynamics of disease spread. Subtyping thereby advances the development of effective containment strategies and, potentially, therapeutic and vaccine strategies. However, identifying viral subtypes in real-time is challenging: SARS-CoV-2 is a novel virus, and the pandemic is rapidly expanding. Viral subtypes may be difficult to detect due to rapid evolution; founder effects are more significant than selection pressure; and the clustering threshold for subtyping is not standardized. We propose to identify mutational signatures of available SARS-CoV-2 sequences using a population-based approach: an entropy measure followed by frequency analysis. These signatures, Informative Subtype Markers (ISMs), define a compact set of nucleotide sites that characterize the most variable (and thus most informative) positions in the viral genomes sequenced from different individuals. Through ISM compression, we find that certain distant nucleotide variants covary, including non-coding and ORF1ab sites covarying with the D614G spike protein mutation which has become increasingly prevalent as the pandemic has spread. ISMs are also useful for downstream analyses, such as spatiotemporal visualization of viral dynamics. By analyzing sequence data available in the GISAID database, we validate the utility of ISM-based subtyping by comparing spatiotemporal analyses using ISMs to epidemiological studies of viral transmission in Asia, Europe, and the United States. In addition, we show the relationship of ISMs to phylogenetic reconstructions of SARS-CoV-2 evolution, and therefore, ISMs can play an important complementary role to phylogenetic tree-based analysis, such as is done in the Nextstrain [1] project. The developed pipeline dynamically generates ISMs for newly added SARS-CoV-2 sequences and updates the visualization of pandemic spatiotemporal dynamics, and is available on Github at https://github.com/EESI/ISM and via an interactive website at https://covid19-ism.coe.drexel.edu/. Author Summary The novel coronavirus responsible for COVID-19, SARS-CoV-2, expanded to reportedly 8.7 million confirmed cases worldwide by June 21, 2020. The global SARS-CoV-2 pandemic highlights the importance of tracking viral transmission dynamics in real-time. Through June 2020, researchers have obtained genetic sequences of SARS-CoV-2 from over 47,000 samples from infected individuals worldwide. Since the virus readily mutates, each sequence of an infected individual contains useful information linked to the individual’s exposure location and sample date. But, there are over 30,000 bases in the full SARS-CoV-2 genome—so tracking genetic variants on a whole-sequence basis becomes unwieldy. We describe a method to instead efficiently identify and label genetic variants, or “subtypes” of SARS-CoV-2. Applying this method results in a compact, 11 base-long compressed label, called an Informative Subtype Marker or “ISM”. We define viral subtypes for each ISM, and show how regional distribution of subtypes track the progress of the pandemic. Major findings include (1) covarying nucleotides with the spike protein which has spread rapidly and (2) tracking emergence of a local subtype across the United States connected to Asia and distinct from the outbreak in New York, which is found to be connected to Europe.

negative: Evolution of genomes, host shifts and the geographic spread of SARS‐CoV and related coronaviruses Severe acute respiratory syndrome (SARS) is a novel human illness caused by a previously unrecognized coronavirus (CoV) termed SARS‐CoV. There are conflicting reports on the animal reservoir of SARS‐CoV. Many of the groups that argue carnivores are the original reservoir of SARS‐CoV use a phylogeny to support their argument. However, the phylogenies in these studies often lack outgroup and rooting criteria necessary to determine the origins of SARS‐CoV. Recently, SARS‐CoV has been isolated from various species of Chiroptera from China (e.g., Rhinolophus sinicus) thus leading to reconsideration of the original reservoir of SARS‐CoV. We evaluated the hypothesis that SARS‐CoV isolated from Chiroptera are the original zoonotic source for SARS‐CoV by sampling SARS‐CoV and non‐SARS‐CoV from diverse hosts including Chiroptera, as well as carnivores, artiodactyls, rodents, birds and humans. Regardless of alignment parameters, optimality criteria, or isolate sampling, the resulting phylogenies clearly show that the SARS‐CoV was transmitted to small carnivores well after the epidemic of SARS in humans that began in late 2002. The SARS‐CoV isolates from small carnivores in Shenzhen markets form a terminal clade that emerged recently from within the radiation of human SARS‐CoV. There is evidence of subsequent exchange of SARS‐CoV between humans and carnivores. In addition SARS‐CoV was transmitted independently from humans to farmed pigs (Sus scrofa). The position of SARS‐CoV isolates from Chiroptera are basal to the SARS‐CoV clade isolated from humans and carnivores. Although sequence data indicate that Chiroptera are a good candidate for the original reservoir of SARS‐CoV, the structural biology of the spike protein of SARS‐CoV isolated from Chiroptera suggests that these viruses are not able to interact with the human variant of the receptor of SARS‐CoV, angiotensin‐converting enzyme 2 (ACE2). In SARS‐CoV we study, both visually and statistically, labile genomic fragments and, putative key mutations of the spike protein that may be associated with host shifts. We display host shifts and candidate mutations on trees projected in virtual globes depicting the spread of SARS‐CoV. These results suggest that more sampling of coronaviruses from diverse hosts, especially Chiroptera, carnivores and primates, will be required to understand the genomic and biochemical evolution of coronaviruses, including SARS‐CoV. © The Willi Hennig Society 2008.

negative: Molecular characterization and phylogenetic analysis of the genome of porcine torovirus In this study, we amplified and sequenced the first genome of porcine torovirus (PToV SH1 strain). The genome was found to be 28,301 bp in length, sharing 79 % identity with Breda virus. It mainly consists of replicase (20,906 bp) and structural genes: spike (4,722 bp), membrane (702 bp), hemagglutinin-esterase (1,284 bp), and nucleocapsid (492 bp). Sequence alignments and structure prediction suggest genetic differences among toroviruses, mainly in NSP1 (papain-like cysteine proteinase domain). Rooted phylogenetic trees were constructed based on the 3C-like proteinase and RNA-dependent RNA polymerase genes. PToV, Berne virus and Breda virus were clustered together, forming a separate branch from white bream virus that was distant from that of the coronaviruses.

negative: Complemented Palindromic Small RNAs First Discovered from SARS Coronavirus In this study, we report for the first time the existence of complemented palindromic small RNAs (cpsRNAs) and propose that cpsRNAs and palindromic small RNAs (psRNAs) constitute a novel class of small RNAs. The first discovered 19-nt cpsRNA UUAACAAGCUUGUUAAAGA, named SARS-CoV-cpsR-19, was detected from a 22-bp DNA complemented palindrome TCTTTAACAAGCTTGTTAAAGA in the severe acute respiratory syndrome coronavirus (SARS-CoV) genome. The phylogenetic analysis supported that this DNA complemented palindrome originated from bat betacoronavirus. The results of RNA interference (RNAi) experiments showed that one 19-nt segment corresponding to SARS-CoV-cpsR-19 significantly induced cell apoptosis. Using this joint analysis of the molecular function and phylogeny, our results suggested that SARS-CoV-cpsR-19 could play a role in SARS-CoV infection or pathogenesis. The discovery of cpsRNAs has paved a way to find novel markers for pathogen detection and to reveal the mechanisms underlying infection or pathogenesis from a different point of view. Researchers can use cpsRNAs to study the infection or pathogenesis of pathogenic viruses when these viruses are not available. The discovery of psRNAs and cpsRNAs, as a novel class of small RNAs, also inspire researchers to investigate DNA palindromes and DNA complemented palindromes with lengths of psRNAs and cpsRNAs in viral genomes.

negative: Evolutionary Dynamics And Geographic Dispersal Of Beta Coronaviruses In African Bats Bats have been shown to serve as reserviour host of various viral agents including coronaviruses. They have also been associated with the novel coronavirus SARS-CoV-2. This has made them an all important agent for CoV evolution and transmission. Our objective in this study was to investigate the dispersal, phylogenomics and evolution of betacoronaviruses (β-CoV) among African bats. Three data sets were generated from sequences of the partial RNA depedent RNA polymerase (RdRP) gene of Bat coronaviruses from seven African, three Asian, and four European countries. Phylogenetic trees was infered using MEGA Vs 7, Phylogeography and Evolutionary analysis were carried out, using Markov Chain Monte Carlo (MCMC) implemented in BEAST, and visualised with SPREAD3. Results showed that majority of the African strains fell within lineage D. Evololutionary rate of the African Bt-β-CoV was 1.301 × 10-3, HPD (1.064 × 10-3 – 1.434 × 10-3), the MCC tree of the African strains showed two clades, lineage D, and lineage C. The TMRCA for lineage D strains was 1972, 95% HPD (1951-1990), and 2010, 95% HPD (2006-2014) for the South African strains. There were inter-continental dispersal of Bt-CoV from China and Hong Kong into Central and Southern Africa. In conclusion we advocate for broader studies of whole genome sequences of BtCoV to further understand the drivers for their emergence and zoonotic spillovers into human population.

negative: Perspectives on therapeutic neutralizing antibodies against the Novel Coronavirus SARS-CoV-2 A newly identified novel coronavirus (SARS-CoV-2) is causing pneumonia-associated respiratory syndrome across the world. Epidemiology, genomics, and pathogenesis of the SARS-CoV-2 show high homology with that of SARS-CoV. Current efforts are focusing on development of specific antiviral drugs. Therapeutic neutralizing antibodies (NAbs) against SARS-CoV-2 will be greatly important therapeutic agents for the treatment of coronavirus disease 2019 (COVID-19). Herein, the host immune responses against SARS-CoV discussed in this review provide implications for developing NAbs and understanding clinical interventions against SARS-CoV-2. Further, we describe the benefits, challenges and considerations of NAbs against SARS-CoV-2. Although many challenges exist, NAbs still offer a therapeutic option to control the current pandemic and the possible re-emergence of the virus in the future, and their development therefore remains a high priority.

negative: Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2;initially named as 2019-nCoV) is responsible for the recent COVID-19 pandemic and polymerase chain reaction (PCR) is the current standard method for its diagnosis from patient samples This study conducted a reassessment of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), through the evaluation of mismatches with publicly available viral sequences An exhaustive evaluation of the sequence variability within the primer/probe target regions of the viral genome was performed using more than 17 000 viral sequences from around the world The analysis showed the presence of mutations/mismatches in primer/probe binding regions of 7 assays out of 27 assays studied A comprehensive bioinformatics approach for in silico inclusivity evaluation of PCR diagnostic assays of SARS-CoV-2 was validated using freely available software programs that can be applied to any diagnostic assay of choice These findings provide potentially important information for clinicians, laboratory professionals and policy-makers

negative: Master Regulator Analysis of the SARS-CoV-2/Human interactome the recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 and causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. In this paper, we set out to shed light on the SARS-CoV-2/host receptor recognition, a crucial factor for successful virus infection. Based on the current knowledge of the interactome between SARS-CoV-2 and host cell proteins, we performed Master Regulator Analysis to detect which parts of the human interactome are most affected by the infection. We detected, amongst others, affected apoptotic and mitochondrial mechanisms, and a downregulation of the ACE2 protein receptor, notions that can be used to develop specific therapies against this new virus.

negative: New insights into the evolutionary features of viral overlapping genes by discriminant analysis Abstract Overlapping genes originate by a mechanism of overprinting, in which nucleotide substitutions in a pre-existing frame induce the expression of a de novo protein from an alternative frame. In this study, I assembled a dataset of 319 viral overlapping genes, which included 82 overlaps whose expression is experimentally known and the respective 237 homologs. Principal component analysis revealed that overlapping genes have a common pattern of nucleotide and amino acid composition. Discriminant analysis separated overlapping from non-overlapping genes with an accuracy of 97%. When applied to overlapping genes with known genealogy, it separated ancestral from de novo frames with an accuracy close to 100%. This high discriminant power was crucial to computationally design variants of de novo viral proteins known to possess selective anticancer toxicity (apoptin) or protection against neurodegeneration (X protein), as well as to detect two new potential overlapping genes in the genome of the new coronavirus SARS-CoV-2.

negative: CoV Genome Tracker: tracing genomic footprints of Covid-19 pandemic Summary Genome sequences constitute the primary evidence on the origin and spread of the 2019-2020 Covid-19 pandemic. Rapid comparative analysis of coronavirus SARS-CoV-2 genomes is critical for disease control, outbreak forecasting, and developing clinical interventions. CoV Genome Tracker is a web portal dedicated to trace Covid-19 outbreaks in real time using a haplotype network, an accurate and scalable representation of genomic changes in a rapidly evolving population. We resolve the direction of mutations by using a bat-associated genome as outgroup. At a broader evolutionary time scale, a companion browser provides gene-by-gene and codon-by-codon evolutionary rates to facilitate the search for molecular targets of clinical interventions. Availability and Implementation CoV Genome Tracker is publicly available at http://cov.genometracker.org and updated weekly with the data downloaded from GISAID (http://gisaid.org). The website is implemented with a custom JavaScript script based on jQuery (https://jquery.com) and D3-force (https://github.com/d3/d3-force). Contact weigang@genectr.hunter.cuny.edu, City University of New York, Hunter College Supplementary Information All supporting scripts developed in JavaScript, Python, BASH, and PERL programming languages are available as Open Source at the GitHub repository https://github.com/weigangq/cov-browser.

negative: [Analysis on the SARS-CoV genome of PUMC01 isolate]. OBJECTIVE To perform variation and phylogenetics analysis on the SARS-CoV genome sequence (PUMC01) isolated in the Peking Union Medical College Hospital. METHODS The cDNA library of SARS-CoV (PUMC01 isolate) was constructed by means of random-priming strategy. Random selected plasmid was sequenced and the genome sequence of SARS-CoV-PUMC01 was assembled by conventional methods (The Genebank Accession No. of SARS-CoV-PUMC01 is AY350750). The variation and phylogenetics analysis were performed by comparing the PUMC01 sequence with other SARS-CoV isolates. RESULTS Ten variation sites were found by comparing PUMC01 isolate with Tor2 and Urbani isolates. In phylogenetic analysis of 18 SARS-CoV isolates, two classes were observed and there is different differential time between these two classes and the different isolates in each class. CONCLUSIONS The evidence of phylogenetic analysis of different SARS-CoV isolates from different region is instructive for understanding the clinical relations between the different isolates and the transmission chain of SARS-CoV.

negative: Sequence analysis of the S1 glycoprotein gene of infectious bronchitis viruses: identification of a novel phylogenetic group in Korea Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.

negative: Growth, detection, quantification, and inactivation of SARS-CoV-2 Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak but disappeared rapidly. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and 5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.

negative: Discovery and Genomic Characterization of a Novel Bat Sapovirus with Unusual Genomic Features and Phylogenetic Position Sapovirus is a genus of caliciviruses that are known to cause enteric disease in humans and animals. There is considerable genetic diversity among the sapoviruses, which are classified into different genogroups based on phylogenetic analysis of the full-length capsid protein sequence. While several mammalian species, including humans, pigs, minks, and dogs, have been identified as animal hosts for sapoviruses, there were no reports of sapoviruses in bats in spite of their biological diversity. In this report, we present the results of a targeted surveillance study in different bat species in Hong Kong. Five of the 321 specimens from the bat species, Hipposideros pomona, were found to be positive for sapoviruses by RT-PCR. Complete or nearly full-length genome sequences of approximately 7.7 kb in length were obtained for three strains, which showed similar organization of the genome compared to other sapoviruses. Interestingly, they possess many genomic features atypical of most sapoviruses, like high G+C content and minimal CpG suppression. Phylogenetic analysis of the viral proteins suggested that the bat sapovirus descended from an ancestral sapovirus lineage and is most closely related to the porcine sapoviruses. Codon usage analysis showed that the bat sapovirus genome has greater codon usage bias relative to other sapovirus genomes. In summary, we report the discovery and genomic characterization of the first bat calicivirus, which appears to have evolved under different conditions after early divergence from other sapovirus lineages.

negative: Phylogenetic tracking of current porcine epidemic diarrhea virus (PEDV) strains in the Philippines To trace the possible route of introduction of porcine epidemic diarrhea virus (PEDV), the phylogenetic relationships of PEDV strains in the regions of epidemicity in the Philippines to PEDV strains that are endemic in other countries were investigated. Partial nucleotide sequences of the S1 spike gene was determined from the PEDV-positive samples and compared with S1 sequences from other countries. Phylogenetic analysis indicated that PEDV strains in the Philippines segregate into two groups. Members of group 1 are related to strains from the USA, Taiwan, Japan and Canada, while those in group 2 are related to strains from China and Vietnam.

negative: Close relationship between SARS-coronavirus and group 2 coronavirus. The sudden appearance and potential lethality of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in humans has resulted in a focusing of new attention on the determination of both its origins and evolution. The relationship existing between SARS-CoV and other groups of coronaviruses was determined via analyses of phylogenetic trees and comparative genomic analyses of the coronavirus genes: polymerase (Orf1ab), spike (S), envelope (E), membrane (M) and nucleocapsid (N). Although the coronaviruses are traditionally classed into 3 groups, with SARS-CoV forming a 4th group, the phylogenetic position and origins of SARS-CoV remain a matter of some controversy. Thus, we conducted extensive phylogenetic analyses of the genes common to all coronavirus groups, using the Neighbor-joining, Maximum-likelihood, and Bayesian methods. Our data evidenced largely identical topology for all of the obtained phylogenetic trees, thus supporting the hypothesis that the relationship existing between SARS-CoV and group 2 coronavirus is a monophyletic one. Additional comparative genomic studies, including sequence similarity and protein secondary structure analyses, suggested that SARS-CoV may bear a closer relationship with group 2 than with the other coronavirus groups. Although our data strongly suggest that group 2 coronaviruses are most closely related with SARS-CoV, further and more detailed analyses may provide us with an increased amount of information regarding the origins and evolution of the coronaviruses, most notably SARS-CoV.

negative: The Prediction of miRNAs in SARS-CoV-2 Genomes: hsa-miR Databases Identify 7 Key miRs Linked to Host Responses and Virus Pathogenicity-Related KEGG Pathways Significant for Comorbidities Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a member of the betacoronavirus family, which causes COVID-19 disease. SARS-CoV-2 pathogenicity in humans leads to increased mortality rates due to alterations of significant pathways, including some resulting in exacerbated inflammatory responses linked to the "cytokine storm" and extensive lung pathology, as well as being linked to a number of comorbidities. Our current study compared five SARS-CoV-2 sequences from different geographical regions to those from SARS, MERS and two cold viruses, OC43 and 229E, to identify the presence of miR-like sequences. We identified seven key miRs, which highlight considerable differences between the SARS-CoV-2 sequences, compared with the other viruses. The level of conservation between the five SARS-CoV-2 sequences was identical but poor compared with the other sequences, with SARS showing the highest degree of conservation. This decrease in similarity could result in reduced levels of transcriptional control, as well as a change in the physiological effect of the virus and associated host-pathogen responses. MERS and the milder symptom viruses showed greater differences and even significant sequence gaps. This divergence away from the SARS-CoV-2 sequences broadly mirrors the phylogenetic relationships obtained from the whole-genome alignments. Therefore, patterns of mutation, occurring during sequence divergence from the longer established human viruses to the more recent ones, may have led to the emergence of sequence motifs that can be related directly to the pathogenicity of SARS-CoV-2. Importantly, we identified 7 key-microRNAs (miRs 8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) with significant links to KEGG pathways linked to viral pathogenicity and host responses. According to Bioproject data (PRJNA615032), SARS-CoV-2 mediated transcriptomic alterations were similar to the target pathways of the selected 7 miRs identified in our study. This mechanism could have considerable significance in determining the symptom spectrum of future potential pandemics. KEGG pathway analysis revealed a number of critical pathways linked to the seven identified miRs that may provide insight into the interplay between the virus and comorbidities. Based on our reported findings, miRNAs may constitute potential and effective therapeutic approaches in COVID-19 and its pathological consequences.

negative: [Further analysis and study based on a visualized method for SARS RNA sequences]. This paper proposed a new kind of visualized method of genome. Using cellular automation theory, the visual method transfers one-dimensional RNA sequence into two-demension visual image. Applying this method to SARS RNA sequence analysis, the characteristic of SARS-CoV differing from Non-SARS is discovered. This paper extracts characteristic genome fragment, visualize them, and study them with some pattern recognition method such as PCA and SVM. The result shows that the characteristic of SARS-CoV is classifiable. Some combined methods can use the characteristic more sufficient as an un-routine method.

negative: Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods SARS-CoV-2 has caused tens of thousands of infections and more than one thousand deaths. There are currently no registered therapies for treating coronavirus infections. Because of time consuming process of new drug development, drug repositioning may be the only solution to the epidemic of sudden infectious diseases. We systematically analyzed all the proteins encoded by SARS-CoV-2 genes, compared them with proteins from other coronaviruses, predicted their structures, and built 19 structures that could be done by homology modeling. By performing target-based virtual ligand screening, a total of 21 targets (including two human targets) were screened against compound libraries including ZINC drug database and our own database of natural products. Structure and screening results of important targets such as 3-chymotrypsin-like protease (3CLpro), Spike, RNA-dependent RNA polymerase (RdRp), and papain like protease (PLpro) were discussed in detail. In addition, a database of 78 commonly used anti-viral drugs including those currently on the market and undergoing clinical trials for SARS-CoV-2 was constructed. Possible targets of these compounds and potential drugs acting on a certain target were predicted. This study will provide new lead compounds and targets for further in vitro and in vivo studies of SARS-CoV-2, new insights for those drugs currently ongoing clinical studies, and also possible new strategies for drug repositioning to treat SARS-CoV-2 infections.

negative: Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China. UNLABELLED Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. IMPORTANCE Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province.

negative: Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

negative: Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Jin Yin-tan Hospital of Wuhan, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

negative: The origin and underlying driving forces of the SARS-CoV-2 outbreak The spread of SARS-CoV-2 since December 2019 has become a pandemic and impacted many aspects of human society. Here, we analyzed genetic variation of SARS-CoV-2 and its related coronavirus and found the evidence of intergenomic recombination. After correction for mutational bias, analysis of 137 SARS-CoV-2 genomes as of 2/23/2020 revealed the excess of low frequency mutations on both synonymous and nonsynonymous sites which is consistent with recent origin of the virus. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China was two time higher than those derived from the rest of the world. In addition, in network analysis, haplotypes collected from Wuhan city were at interior and have more mutational connections, both of which are consistent with the observation that the outbreak of cov-19 was originated from China. SUMMARY In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated.

negative: A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2 Effective countermeasures against the recent emergence and rapid expansion of the 2019 novel coronavirus (SARS-CoV-2) require the development of data and tools to understand and monitor its spread and immune responses to it. However, little information is available about the targets of immune responses to SARS-CoV-2. We used the Immune Epitope Database and Analysis Resource (IEDB) to catalog available data related to other coronaviruses. This includes SARS-CoV, which has high sequence similarity to SARS-CoV-2 and is the best-characterized coronavirus in terms of epitope responses. We identified multiple specific regions in SARS-CoV-2 that have high homology to the SARS-CoV virus. Parallel bioinformatic predictions identified a priori potential B and T cell epitopes for SARS-CoV-2. The independent identification of the same regions using two approaches reflects the high probability that these regions are promising targets for immune recognition of SARS-CoV-2. These predictions can facilitate effective vaccine design against this virus of high priority.

negative: Drug Development and Medicinal Chemistry Efforts toward SARS‐Coronavirus and Covid‐19 Therapeutics The COVID‐19 pandemic caused by SARS‐CoV‐2 infection is spreading at an alarming rate and has created an unprecedented health emergency around the globe. There is no effective vaccine or approved drug treatment against COVID‐19 and other pathogenic coronaviruses. The development of antiviral agents is an urgent priority. Biochemical events critical to the coronavirus replication cycle provided a number of attractive targets for drug development. These include, spike protein for binding to host cell‐surface receptors, proteolytic enzymes that are essential for processing polyproteins into mature viruses, and RNA‐dependent RNA polymerase for RNA replication. There has been a lot of ground work for drug discovery and development against these targets. Also, high‐throughput screening efforts have led to the identification of diverse lead structures, including natural product‐derived molecules. This review highlights past and present drug discovery and medicinal‐chemistry approaches against SARS‐CoV, MERS‐CoV and COVID‐19 targets. The review hopes to stimulate further research and will be a useful guide to the development of effective therapies against COVID‐19 and other pathogenic coronaviruses.

negative: Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases, which has a 5′ cap structure and 3′ polyadenylation tract. The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames: 5′ -orf1a-orf1ab-s-3a-3b-e-m-6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Beijing isolate is lone virus in group III and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.

negative: Recombination and lineage-specific mutations led to the emergence of SARS-CoV-2 The recent outbreak of a new coronavirus (SARS-CoV-2) in Wuhan, China, underscores the need for understanding the evolutionary processes that drive the emergence and adaptation of zoonotic viruses in humans. Here, we show that recombination in betacoronaviruses, including human-infecting viruses like SARS-CoV and MERS-CoV, frequently encompasses the Receptor Binding Domain (RBD) in the Spike gene. We find that this common process likely led to a recombination event at least 11 years ago in an ancestor of the SARS-CoV-2 involving the RBD. As a result of this recombination event, SARS-CoV and SARS-CoV-2 share a similar genotype in RBD, including two insertions (positions 432-436 and 460-472), and alleles 427N and 436Y. Both 427N and 436Y belong to a helix that interacts with the human ACE2 receptor. Ancestral state analyses revealed that SARS-CoV-2 differentiated from its most recent common ancestor with RaTG13 by accumulating a significant number of amino acid changes in the RBD. In sum, we propose a two-hit scenario in the emergence of the SARS-CoV-2 virus whereby the SARS-CoV-2 ancestors in bats first acquired genetic characteristics of SARS-CoV by incorporation of a SARS-like RBD through recombination before 2009, and subsequently, the lineage that led to SARS-CoV-2 accumulated further, unique changes specifically in the RBD.

negative: Chapter Three The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing Abstract Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26–32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7–nsp10) act as crucial cofactors of these enzymes and contribute to the emerging “nsp interactome.” Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.

negative: Genomic characterization of seven distinct bat coronaviruses in Kenya Abstract To better understand the genetic diversity and genomic features of 41 coronaviruses (CoVs) identified from Kenya bats in 2006, seven CoVs as representatives of seven different phylogenetic groups identified from partial polymerase gene sequences, were subjected to extensive genomic sequencing. As a result, 15–16kb nucleotide sequences encoding complete RNA dependent RNA polymerase, spike, envelope, membrane, and nucleocapsid proteins plus other open reading frames (ORFs) were generated. Sequences analysis confirmed that the CoVs from Kenya bats are divergent members of Alphacoronavirus and Betacoronavirus genera. Furthermore, the CoVs BtKY22, BtKY41, and BtKY43 in Alphacoronavirus genus and BtKY24 in Betacoronavirus genus are likely representatives of 4 novel CoV species. BtKY27 and BtKY33 are members of the established bat CoV species in Alphacoronavirus genus and BtKY06 is a member of the established bat CoV species in Betacoronavirus genus. The genome organization of these seven CoVs is similar to other known CoVs from the same groups except for differences in the number of putative ORFs following the N gene. The present results confirm a significant diversity of CoVs circulating in Kenya bats. These Kenya bat CoVs are phylogenetically distant from any previously described human and animal CoVs. However, because of the examples of host switching among CoVs after relatively minor sequence changes in S1 domain of spike protein, a further surveillance in animal reservoirs and understanding the interface between host susceptibility is critical for predicting and preventing the potential threat of bat CoVs to public health.

negative: The revolution of the biology of the genome Sequence data of entire eukaryotic genomes and their detailed comparison have provided new evidence on genome evolution. The major mechanisms involved in the increase of genome sizes are polyploidization and gene duplication. Subsequent gene silencing or mutations, preferentially in regulatory sequences of genes, modify the genome and permit the development of genes with new properties. Mechanisms such as lateral gene transfer, exon shuffling or the creation of new genes by transposition contribute to the evolution of a genome, but remain of relatively restricted relevance. Mechanisms to decrease genome sizes and, in particular, to remove specific DNA sequences, such as blocks of satellite DNAs, appear to involve the action of RNA interference (RNAi). RNAi mechanisms have been proven to be involved in chromatin packaging related with gene inactivation as well as in DNA excision during the macronucleus development in ciliates.

negative: Bioinformatical study on the proteomics and evolution of SARS-CoV A novel coronavirus has been identified as the causative agent of the severe acute respiratory syndrome (SARS). For all the SARS-CoV associated proteins derivated from the SARS-CoV genome, the physiochemical properties such as the molecular weight, isoelectric point and extinction coefficient of each protein were calculated. The transmembrane segments and subcellular localization (SubLocation) prediction and conserved protein motifs search against database were employed to analyze the function of SARS-CoV proteins. Also, the homology protein sequence alignment and evolutionary distance matrix calculation between SARS-CoV associated proteins and the corresponding proteins of other coronaviruses were employed to identify the classification and phylogenetic relationship between SARS-CoV and other coronaviruses. The results showed that SARS-CoV is a novel coronavirus which is different from any of the three previously known groups of coronviruses, but it is closer to Bo-CoV and MHV than to other coronaviruses. This study is in aid of experimental determination of SARS-CoV proteomics and the development of antiviral vaccine.

negative: Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 Abstract SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 52,020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

negative: A Novel Approach to Clustering Genome Sequences Using Inter-nucleotide Covariance Classification of DNA sequences is an important issue in the bioinformatics study, yet most existing methods for phylogenetic analysis including Multiple Sequence Alignment (MSA) are time-consuming and computationally expensive. The alignment-free methods are popular nowadays, whereas the manual intervention in those methods usually decreases the accuracy. Also, the interactions among nucleotides are neglected in most methods. Here we propose a new Accumulated Natural Vector (ANV) method which represents each DNA sequence by a point in ℝ(18). By calculating the Accumulated Indicator Functions of nucleotides, we can further find an Accumulated Natural Vector for each sequence. This new Accumulated Natural Vector not only can capture the distribution of each nucleotide, but also provide the covariance among nucleotides. Thus global comparison of DNA sequences or genomes can be done easily in ℝ(18). The tests of ANV of datasets of different sizes and types have proved the accuracy and time-efficiency of the new proposed ANV method.

negative: Bovine Kobuvirus in Calves with Diarrhea, United States We detected bovine kobuvirus (BKV) in calves with diarrhea in the United States. The strain identified is related genetically to BKVs detected in other countries. Histopathologic findings also confirmed viral infection in 2 BKV cases. Our data show BKV is a potential causative agent for diarrhea in calves.

negative: Discovery and Prevalence of Divergent RNA Viruses in European Field Voles and Rabbits The advent of unbiased metagenomic virus discovery has revolutionized studies of virus biodiversity and evolution. Despite this, our knowledge of the virosphere, including in mammalian species, remains limited. We used unbiased metagenomic sequencing to identify RNA viruses in European field voles and rabbits. Accordingly, we identified a number of novel RNA viruses including astrovirus, rotavirus A, picorna-like virus and a narmovirus (paramyxovirus). In addition, we identified a sobemovirus and a novel luteovirus that likely originated from the rabbit diet. These newly discovered viruses were often divergent from those previously described. The novel astrovirus was most closely related to a virus sampled from the rodent-eating European roller bird (Coracias garrulous). PCR screening revealed that the novel narmovirus in the UK field vole had a prevalence of approximately 4%, and shared common ancestry with other rodent narmoviruses sampled globally. Two novel rotavirus A sequences were detected in a UK field vole and a French rabbit, the latter with a prevalence of 5%. Finally, a highly divergent picorna-like virus found in the gut of the French rabbit virus was only ~35% similar to an arilivirus at the amino acid level, suggesting the presence of a novel viral genus within the Picornaviridae.

negative: Accurate Identification of SARS-CoV-2 from Viral Genome Sequences using Deep Learning One of the reasons for the fast spread of SARS-CoV-2 is the lack of accuracy in detection tools in the clinical field. Molecular techniques, such as quantitative real-time RT-PCR and nucleic acid sequencing methods, are widely used to identify pathogens. For this particular virus, however, they have an overall unsatisfying detection rate, due to its relatively recent emergence and still not completely understood features. In addition, SARS-CoV-2 is remarkably similar to other Coronaviruses, and it can present with other respiratory infections, making identification even harder. To tackle this issue, we propose an assisted detection test, combining molecular testing with deep learning. The proposed approach employs a state-of-the-art deep convolutional neural network, able to automatically create features starting from the genome sequence of the virus. Experiments on data from the Novel Coronavirus Resource (2019nCoVR) show that the proposed approach is able to correctly classify SARS-CoV-2, distinguishing it from other coronavirus strains, such as MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-229E, HCoV-HKU1, and SARS-CoV regardless of missing information and errors in sequencing (noise). From a dataset of 553 complete genome non-repeated sequences that vary from 1,260 to 31,029 bps in length, the proposed approach classifies the different coronaviruses with an average accuracy of 98.75% in a 10-fold cross-validation, identifying SARS-CoV-2 with an AUC of 98%, specificity of 0.9939 and sensitivity of 1.00 in a binary classification. Then, using the same basis, we classify SARS-CoV-2 from 384 complete viral genome sequences with human host, that contain the gene ORF1ab from the NCBI with a 10-fold accuracy of 98.17%, a specificity of 0.9797 and sensitivity of 1.00. Furthermore, an in-depth analysis of the results allow us to identify base pairs sequences that are unique to SARS-CoV-2 and do not appear in other virus strains, that could then be used as a base for designing new primers and examined by experts to extract further insights. These preliminary results seem encouraging enough to identify deep learning as a promising research venue to develop assisted detection tests for SARS-CoV-2. At this end the interaction between viromics and deep learning, will hopefully help to solve global infection problems. In addition, we offer our code and processed data to be used for diagnostic purposes by medical doctors, virologists and scientists involved in solving the SARS-CoV-2 pandemic. As more data become available we will update our system.

negative: Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.

negative: The early diversification of influenza A/H1N1pdm Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification. Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 – July 9, 2009, including relatively large samples from the US states of Wisconsin and New York. Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures. Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns.

negative: Genetic diversity and evolution of SARS-CoV-2 COVID-19 is a viral respiratory illness caused by a new coronavirus called SARS-CoV-2. The World Health Organization declared the SARS-CoV-2 outbreak a global public health emergency. We performed genetic analyses of eighty-six complete or near-complete genomes of SARS-CoV-2 and revealed many mutations and deletions on coding and non-coding regions. These observations provided evidence of the genetic diversity and rapid evolution of this novel coronavirus.

negative: Relating phylogenetic trees to transmission trees of infectious disease outbreaks. Transmission events are the fundamental building blocks of the dynamics of any infectious disease. Much about the epidemiology of a disease can be learned when these individual transmission events are known or can be estimated. Such estimations are difficult and generally feasible only when detailed epidemiological data are available. The genealogy estimated from genetic sequences of sampled pathogens is another rich source of information on transmission history. Optimal inference of transmission events calls for the combination of genetic data and epidemiological data into one joint analysis. A key difficulty is that the transmission tree, which describes the transmission events between infected hosts, differs from the phylogenetic tree, which describes the ancestral relationships between pathogens sampled from these hosts. The trees differ both in timing of the internal nodes and in topology. These differences become more pronounced when a higher fraction of infected hosts is sampled. We show how the phylogenetic tree of sampled pathogens is related to the transmission tree of an outbreak of an infectious disease, by the within-host dynamics of pathogens. We provide a statistical framework to infer key epidemiological and mutational parameters by simultaneously estimating the phylogenetic tree and the transmission tree. We test the approach using simulations and illustrate its use on an outbreak of foot-and-mouth disease. The approach unifies existing methods in the emerging field of phylodynamics with transmission tree reconstruction methods that are used in infectious disease epidemiology.

negative: Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient’s sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

negative: Detection and phylogenetic characterization of astroviruses in insectivorous bats from Central‐Southern Italy In recent years, bats have been found to harbour many viruses, raising several questions about their role as reservoirs and potential disseminators of zoonotic viruses. We investigated the presence of six virus families in bats in three regions of Central‐Southern Italy. Astroviruses were identified in seven of 13 bat species. Sequence analysis revealed marked genetic heterogeneity among the astroviruses identified, with nucleotide identity ranging between 60.26% and 87.62%. Astrovirus diversity was not associated with the bat species, the geographic areas or the bat colony, suggesting the circulation of several astrovirus strains in Italian ecosystems. Genetic diversification and interspecies transmission appear common in bat astroviruses and could provide, potentially, the bases for transmission to humans and other mammals. Yet overemphasizing this risk might have detrimental consequences for bat conservation and preservation of the important ecosystem services bats provide.

negative: Diverse gammacoronaviruses detected in wild birds from Madagascar To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

negative: Large-Scale Phylogenetic Analysis of Emerging Infectious Diseases Microorganisms that cause infectious diseases present critical issues of national security, public health, and economic welfare. For example, in recent years, highly pathogenic strains of avian influenza have emerged in Asia, spread through Eastern Europe, and threaten to become pandemic. As demonstrated by the coordinated response to Severe Acute Respiratory Syndrome (SARS) and influenza, agents of infectious disease are being addressed via large-scale genomic sequencing. The goal of genomic sequencing projects are to rapidly put large amounts of data in the public domain to accelerate research on disease surveillance, treatment, and prevention. However, our ability to derive information from large comparative genomic datasets lags far behind acquisition. Here we review the computational challenges of comparative genomic analyses, specifically sequence alignment and reconstruction of phylogenetic trees. We present novel analytical results on two important infectious diseases, Severe Acute Respiratory Syndrome (SARS) and influenza. SARS and influenza have similarities and important differences both as biological and comparative genomic analysis problems. Influenza viruses (Orthymxyoviridae) are RNA based. Current evidence indicates that influenza viruses originate in aquatic birds from wild populations. Influenza has been studied for decades via well-coordinated international efforts. These efforts center on surveillance via antibody characterization of the hemagglutinin (HA) and neuraminidase (N) proteins of the circulating strains to inform vaccine design. However, we still do not have a clear understanding of (1) various transmission pathways such as the role of intermediate hosts like swine and domestic birds and (2) the key mutation and genomic recombination events that underlie periodic pandemics of influenza. In the past 30 years, sequence data from HA and N loci has become an important data type. In the past year, full genomic data has become prominent. These data present exciting opportunities to address unanswered questions in influenza pandemics. SARS is caused by a previously unrecognized lineage of coronavirus, SARS-CoV, which like influenza has an RNA based genome. Although SARS-CoV is widely believed to have originated in animals, there remains disagreement over the candidate animal source that lead to the original outbreak of SARS. In contrast to the long history of the study of influenza, SARS was only recognized in late 2002 and the virus that causes SARS has been documented primarily by genomic sequencing. In the past, most studies of influenza were performed on a limited number of isolates and genes suited to a particular problem. Major goals in science today are to understand emerging diseases in broad geographic, environmental, societal, biological, and genomic contexts. Synthesizing diverse information brought together by various researchers is important to find out what can be done to prevent future outbreaks [JON03]. Thus comprehensive means to organize and analyze large amounts of diverse information are critical. For example, the relationships of isolates and patterns of genomic change observed in large datasets might not be consistent with hypotheses formed on partial data. Moreover when researchers rely on partial datasets, they restrict the range of possible discoveries. Phylogenetics is well suited to the complex task of understanding emerging infectious disease. Phylogenetic analyses can test many hypotheses by comparing diverse isolates collected from various hosts, environments, and points in time and organizing these data into various evolutionary scenarios. The products of a phylogenetic analysis are a graphical tree of ancestor–descendent relationships and an inferred summary of mutations, recombination events, host shifts, geographic, and temporal spread of the viruses. However, this synthesis comes at a price. The cost of computation of phylogenetic analysis expands combinatorially as the number of isolates considered increases. Thus, large datasets like those currently produced are commonly considered intractable. We address this problem with synergistic development of heuristics tree search strategies and parallel computing.

negative: Data stream dataset of SARS-CoV-2 genome As of May 25, 2020, the novel coronavirus disease (called COVID-19) spread to more than 185 countries/regions with more than 348,000 deaths and more than 5,550,000 confirmed cases. In the bioinformatics area, one of the crucial points is the analysis of the virus nucleotide sequences using approaches such as data stream techniques and algorithms. However, to make feasible this approach, it is necessary to transform the nucleotide sequences string to numerical stream representation. Thus, the dataset provides four kinds of data stream representation (DSR) of SARS-CoV-2 virus nucleotide sequences. The dataset provides the DSR of 1557 instances of SARS-CoV-2 virus, 11540 other instances of other viruses from the Virus-Host DB dataset, and three instances of Riboviria viruses from NCBI (Betacoronavirus RaTG13, bat-SL-CoVZC45, and bat-SL-CoVZXC21).

negative: The divergence between SARS-CoV-2 and RaTG13 might be overestimated due to the extensive RNA modification Aim: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world. There is urgent need to understand the phylogeny, divergence and origin of SARS-CoV-2. Materials & methods: A recent study claimed that there was 17% divergence between SARS-CoV-2 and RaTG13 (a SARS-related coronaviruses) on synonymous sites by using sequence alignment. We re-analyzed the sequences of the two coronaviruses with the same methodology. Results: We found that 87% of the synonymous substitutions between the two coronaviruses could be potentially explained by the RNA modification system in hosts, with 65% contributed by deamination on cytidines (C-T mismatches) and 22% contributed by deamination on adenosines (A-G mismatches). Conclusion: Our results demonstrate that the divergence between SARS-CoV-2 and RaTG13 has been overestimated.

negative: COVID-19: Viral-host interactome analyzed by network based-approach model to study pathogenesis of SARS-CoV-2 infection Background Epidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. Methods We investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV-host interactome was carried out in order to provide a theoretic host-pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein-protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. Results Although the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. Conclusions In this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.

negative: Genetic Analysis Tracks SARS-CoV-2 Mutations in Human Hosts.

negative: Genetic distance of SARS coronavirus from the recent natural case Abstract Phylogenetic analysis of SARS coronavirus isolates based on the spike gene and protein sequence using Neighbor-Joining, maximum likelihood and Bayesian inference methods indicated that a recent human SARS-CoV isolate was closer to some human SARS-CoV isolates from earlier epidemic phase than to the SARS-CoV-like viruses isolated from wild animals during previous epidemic phase. A reasonable judgment based on phylogenetic relationship and sequence variations it is likely that the recent human SARS-CoV isolate is closer to an unknown SARS-CoV predecessor.

negative: SARS‐CoV‐2 and human milk: What is the evidence? The novel coronavirus SARS‐CoV‐2 has emerged as one of the most compelling and concerning public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical and public health communities has rapidly engaged to collectively find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS‐CoV‐2 transmission. Although respiratory droplets are a known mechanism of transmission, other mechanisms are likely. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronaviruses (including SARS‐CoV‐2) via human milk and/or breastfeeding. Results of the literature search reported here (finalized on 17 April 2020) revealed a single study providing some evidence of vertical transmission of human coronavirus 229E; a single study evaluating presence of SARS‐CoV in human milk (it was negative); and no published data on MERS‐CoV and human milk. We identified 13 studies reporting human milk tested for SARS‐CoV‐2; one study (a non‐peer‐reviewed preprint) detected the virus in one milk sample, and another study detected SARS‐CoV‐2 specific IgG in milk. Importantly, none of the studies on coronaviruses and human milk report validation of their collection and analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, SARS‐CoV‐2) during breastfeeding are discussed.

negative: Equine rhinitis B viruses in horse fecal samples from the Middle East BACKGROUND: Among all known picornaviruses, only two species, equine rhinitis A virus and equine rhinitis B virus (ERBV) are known to infect horses, causing respiratory infections. No reports have described the detection of ERBV in fecal samples of horses and no complete genome sequences of ERBV3 are available. METHODS: We performed a molecular epidemiology study to detect ERBVs in horses from Dubai and Hong Kong. Complete genome sequencing of the ERBVs as well as viral loads and genome, phylogenetic and evolutionary analysis were performed on the positive samples. RESULTS: ERBV was detected in four (13.8 %) of the 29 fecal samples in horses from Dubai, with viral loads 8.28 × 10(3) to 5.83 × 10(4) copies per ml, but none of the 47 fecal samples in horses from Hong Kong by RT-PCR. Complete genome sequencing and phylogenetic analysis showed that three of the four strains were ERBV3 and one was ERBV2. The major difference between the genomes of ERBV3 and those of ERBV1 and ERBV2 lied in the amino acid sequences of their VP1 proteins. The Ka/Ks ratios of all the coding regions in the ERBV3 genomes were all <0.1, suggesting that ERBV3 were stably evolving in horses. Using the uncorrelated lognormal distributed relaxed clock model on VP1 gene, the date of the most recent common ancestor (MRCA) of ERBV3 was estimated to be 1785 (HPDs, 1176 to 1937) and the MRCA dates of ERBV1 and ERBV2 were estimated to be 1848 (HPDs, 1466 to 1949) respectively. CONCLUSIONS: Both acid stable (ERBV3) and acid labile (ERBV2) ERBVs could be found in fecal samples of horses. Detection of ERBVs in fecal samples would have implications for their transmission and potential role in gastrointestinal diseases as well as fecal sampling as an alternative method of identifying infected horses.

negative: Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs Abstract The SARS-associated coronavirus (SARS-CoV) main proteinase is a key enzyme in viral polyprotein processing. To allow structure-based design of drugs directed at SARS-CoV main proteinase, we predicted its binding pockets and affinities with existing HIV, psychotic and parasite drugs (lopinavir, ritonavir, niclosamide and promazine), which show signs of inhibiting the replication of SARS-CoV. Our results suggest that these drugs and another two HIV inhibitors (PNU and UC2) could be used as templates for designing SARS-CoV proteinase inhibitors.

negative: Molecular evolutionary analysis of pH1N1 2009 influenza virus in Reunion Island, South West Indian Ocean region: a cohort study. BACKGROUND/OBJECTIVES Molecular epidemiology is a powerful tool to decipher the dynamics of viral transmission, quasispecies temporal evolution and origins. Little is known about the pH1N1 molecular dynamics in general population. A prospective study (CoPanFlu-RUN) was carried out in Reunion Island to characterize pH1N1 genetic variability and molecular evolution occurring in population during the pH1N1 Influenza pandemic in 2009. METHODOLOGY We directly amplified pH1N1 genomes from 28 different nasal swabs (26 individuals from 21 households). Fifteen strains were fully sequenced and 13 partially. This includes pairs of sequences from different members of 5 separate households; and two pairs from individuals, collected at different times. We assessed the molecular evolution of pH1N1 by genetic variability and phylogenetic analyses. PRINCIPAL FINDINGS We found that i) Reunion pH1N1 sequences stemmed from global "clade 7" but shaped two phylogenetic sub-clades; ii) D239E mutation was identified in the hemagglutinin protein of all Reunion sequences, a mutation which has been associated elsewhere with mild-, upper-respiratory tract pH1N1 infecting strains; iii) Date estimates from molecular phylogenies predicted clade emergence some time before the first detection of pH1N1 by the epidemiological surveillance system; iv) Phylogenetic relatedness was observed between Reunion pH1N1 viruses and those from other countries in South-western Indian Ocean area; v) Quasispecies populations were observed within households and individuals of the cohort-study. CONCLUSIONS Surveillance and/or prevention systems presently based on Influenza virus sequence variation should take into account that the majority of studies of pH1N1 Influenza generate genetic data for the HA/NA viral segments obtained from hospitalized-patients, which is potentially non-representative of the overall viral diversity within whole populations. Our observations highlight the importance of collecting unbiased data at the community level and conducting whole genome analysis to accurately understand viral dynamics.

negative: WSE, a new sequence distance measure based on word frequencies Abstract In this article, we present a new distance metric, the Weighted Sequence Entropy (WSE), based on the short word composition of biological sequences. As a revision of the classical relative entropy (RE), our metric (1) works equivalently with RE in the case of small k, (2) avoids the degeneracy when some word types are absent in one sequence but not in the other. Experiments on 25 viruses including SARS-CoVs show that our method and RE give exactly the same phylogenetic tree when word length k ⩽ 3 . When k > 3 , our method still works and gets convergent phylogenetic topology but the RE gives degenerate results.

negative: Coding potential and sequence conservation of SARS-CoV-2 and related animal viruses In December 2019, a novel human-infecting coronavirus (SARS-CoV-2) was recognized in China. In a few months, SARS-CoV-2 has caused thousands of disease cases and deaths in several countries. Phylogenetic analyses indicated that SARS-CoV-2 clusters with SARS-CoV in the Sarbecovirus subgenus and viruses related to SARS-CoV-2 were identified from bats and pangolins. Coronaviruses have long and complex genomes with high plasticity in terms of gene content. To date, the coding potential of SARS-CoV-2 remains partially unknown. We thus used available sequences of bat and pangolin viruses to determine the selective events that shaped the genome structure of SARS-CoV-2 and to assess its coding potential. By searching for signals of significantly reduced variability at synonymous sites (dS), we identified six genomic regions, one of these corresponding to the programmed -1 ribosomal frameshift. The most prominent signal of dS reduction was observed within the E gene. A genome-wide analysis of conserved RNA structures indicated that this region harbors a putative functional RNA element that is shared with the SARS-CoV lineage. Additional signals of reduced dS indicated the presence of internal ORFs. Whereas the presence ORF9a (internal to N) was previously proposed by homology with a well characterized protein of SARS-CoV, ORF3h (for hypothetical, within ORF3a) was not previously described. The predicted product of ORF3h has 90% identity with the corresponding predicted product of SARS-CoV and displays features suggestive of a viroporin. Finally, analysis of the putative ORF10 revealed high dN/dS (3.82) in SARS-CoV-2 and related coronaviruses. In the SARS-CoV lineage, the ORF is predicted to encode a truncated protein and is neutrally evolving. These data suggest that ORF10 encodes a functional protein in SARS-CoV-2 and that positive selection is driving its evolution. Experimental analyses will be necessary to validate and characterize the coding and non-coding functional elements we identified.

negative: Molecular targets for COVID-19 drug development: Enlightening Nigerians about the pandemic and future treatment There is little or no research initiated on enlightening Nigerians about the pathogenesis, targets for drug development, and drug repositioning for SARS-CoV-2 infection. COVID-19 is a viral infection causing symptoms like dry cough, sore throat, nasal congestion, tiredness, fever, loss of taste and smell etc. Th disease was first reported in Wuhan, China, in December 2019. The infection is caused by SARS-CoV-2, which is the third introduction of a highly pathogenic coronavirus into the human population. Coronaviruses are viruses with a positive RNA envelope assigned to α, β, γ, and δ genera. Moreover, SARS-CoV-2 belongs to the β genus. The four structural proteins of β coronavirus are membrane (M), envelope (E), spike (S), and nucleocapsid (N) protein, mediation of coronavirus host infection is established by spike (S) protein. Therefore, the search for drug development targets and repositioning of existing therapeutics is essential for fighting the present pandemic. It was reviewed that therapeutics targeting SARS-CoV-2 binding to ACE2 receptor, viral RNA synthesis and replication, 3CLpro, RdRp, and helicase will play a crucial role in the development of treatment for SARS-CoV-2 infection. Furthermore, the RdRp and spike protein of SARS-CoV-2 are the most promising targets for drug development and repositioning and vaccine development. Remdesivir combination with chloroquine/hydroxychloroquine are promising drug repositioning for the treatment of COVID-19, and mRNA-1273 targeting spike protein is the promising vaccine. However, as patient management and drug repositioning are taking place, it is imperative to identify other promising targets used by SARS-CoV-2 to establish infection, to develop novel therapeutics.

negative: Can FDG PET predict radiation treatment outcome in head and neck cancer? Results of a prospective study. PURPOSE In head and neck cancer (HNC) various treatment strategies have been developed to improve outcome, but selecting patients for these intensified treatments remains difficult. Therefore, identification of novel pretreatment assays to predict outcome is of interest. In HNC there are indications that pretreatment tumour (18)F-fluorodeoxyglucose (FDG) uptake may be an independent prognostic factor. The aim of this study was to assess the prognostic value of FDG uptake and CT-based and FDG PET-based primary tumour volume measurements in patients with HNC treated with (chemo)radiotherapy. METHODS A total of 77 patients with stage II-IV HNC who were eligible for definitive (chemo)radiotherapy underwent coregistered pretreatment CT and FDG PET. The gross tumour volume of the primary tumour was determined on the CT (GTV(CT)) and FDG PET scans. Five PET segmentation methods were applied: interpreting FDG PET visually (PET(VIS)), applying an isocontour at a standardized uptake value (SUV) of 2.5 (PET(2.5)), using fixed thresholds of 40% and 50% (PET(40%), PET(50%)) of the maximum intratumoral FDG activity (SUV(MAX)) and applying an adaptive threshold based on the signal-to-background (PET(SBR)). Mean FDG uptake for each PET-based volume was recorded (SUV(mean)). Subsequently, to determine the metabolic volume, the integrated SUV was calculated as the product of PET-based volume and SUV(mean). All these variables were analysed as potential predictors of local control (LC), regional recurrence-free survival (RRFS), distant metastasis-free survival (DMFS), disease-free survival (DFS) and overall survival (OS). RESULTS In oral cavity/oropharynx tumours PET(VIS) was the only volume-based method able to predict LC. Both PET(VIS) and GTV(CT) were able to predict DMFS, DFS and OS in these subsites. Integrated SUVs were associated with LC, DMFS, DFS and OS, while SUV(mean) and SUV(MAX) were not. In hypopharyngeal/laryngeal tumours none of the variables was associated with outcome. CONCLUSION There is no role yet for pretreatment FDG PET as a predictor of (chemo)radiotherapy outcome in HNC in daily routine. However, this potential application needs further exploration, focusing both on FDG PET-based primary tumour volume, integrated SUV and SUV(MAX) of the primary tumour.

negative: An in silico map of the SARS-CoV-2 RNA Structurome SARS-CoV-2 is a positive-sense single-stranded RNA virus that has exploded throughout the global human population. This pandemic coronavirus strain has taken scientists and public health researchers by surprise and knowledge of its basic biology (e.g. structure/function relationships in its genomic, messenger and template RNAs) and modes for therapeutic intervention lag behind that of other human pathogens. In this report we used a recently-developed bioinformatics approach, ScanFold, to deduce the RNA structural landscape of the SARS-CoV-2 transcriptome. We recapitulate known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that the SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a huge reservoir of potential drug targets for RNA-binding small molecules. Our results also predict regions that are accessible for intermolecular interactions, which can aid in the design of antisense therapeutics. All results are made available via a public database (the RNAStructuromeDB) where they may hopefully drive drug discovery efforts to inhibit SARS-CoV-2 pathogenesis.

negative: Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach The sudden emergence of severe respiratory disease, caused by a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has recently become a public health emergency. Genome sequence analysis of SARS-CoV-2 revealed its close resemblance to the earlier reported SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). However, initial testing of the drugs used against SARS-CoV and MERS-CoV has been ineffective in controlling SARS-CoV-2. The present review looks to highlight the differences in genomic, proteomic, pathogenesis, and therapeutic strategies of SARS-CoV-2. We have carried out sequence analysis of potential drug target proteins in SARS-CoV-2 and, compared them with SARS-CoV-1 and MERS viruses. Analysis of mutations in the coding and non-coding regions, genetic diversity, and pathogenicity of SARS-CoV-2 has also been done. A detailed structural analysis of drug target proteins was performed to gain insights into the mechanism of pathogenesis, structure-function relationships, and the development of structure-guided therapeutic approaches. The cytokine profiling and inflammatory signalling are different in the case of SARS-CoV-2 infection. We also highlighted possible therapies and their mechanism of action followed by clinical manifestation. Our analysis suggests a minimal variation in the genome sequence of SARS-CoV-2, may be responsible for a drastic change in the structures of target proteins, makes available drugs ineffective.

negative: Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection Summary Background The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. Methods We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). Findings We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. Interpretation Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations. Published online May 9, 2003 http://image.thelancet.com/extras/03art4454web.pdf

negative: Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS–coronavirus 2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the infectious disease COVID-19, which was first reported in Wuhan, China in December, 2019. Despite the tremendous efforts to control the disease, COVID-19 has now spread to over 100 countries and caused a global pandemic. SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. Here, we investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. We found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. We found experimentally that cats are susceptible to airborne infection. Our study provides important insights into the animal models for SARS-CoV-2 and animal management for COVID-19 control.

negative: Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019 A novel coronavirus (nCoV-2019) was the cause of an outbreak of respiratory illness detected in Wuhan, Hubei Province, China in December of 2019. Genomic analyses of nCoV-2019 determined a 96% resemblance with a coronavirus isolated from a bat in 2013 (RaTG13); however, the receptor binding motif (RBM) of these two genomes share low sequence similarity. This divergence suggests a possible alternative source for the RBM coding sequence in nCoV-2019. We identified high sequence similarity in the RBM between nCoV-2019 and a coronavirus genome reconstructed from a viral metagenomic dataset from pangolins possibly indicating a more complex origin for nCoV-2019.

negative: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002–2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients’ sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

negative: Spike protein binding prediction with neutralizing antibodies of SARS-CoV-2 Coronavirus disease 2019 (COVID-19) is a new emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV), originated in Wuhan seafood and animal market, China. Since December 2019, more than 69,000 cases of COVID-19 have been confirmed in China and quickly spreads to other counties. Currently, researchers put their best efforts to identify effective drugs for COVID-19. The neutralizing antibody, which binds to viral capsid in a manner that inhibits cellular entry of virus and uncoating of the genome, is the specific defense against viral invaders. In this study, we investigate to identify neutralizing antibodies that can bind to SARS-CoV-2 Sipke (S) protein and interfere with the interaction between viral S protein and a host receptor by bioinformatic methods. The sequence analysis of S protein showed two major differences in the RBD region of the SARS-CoV-2 S protein compared to SARS-CoV and SARS-CoV related bat viruses (btSARS-CoV). The insertion regions were close to interacting residues with the human ACE2 receptor. Epitope analysis of neutralizing antibodies revealed that SARS-CoV neutralizing antibodies used conformational epitopes, whereas MERS-CoV neutralizing antibodies used a common linear epitope region, which contributes to form the β-sheet structure in MERS-CoV S protein and deleted in SARS-CoV-2 S protein. To identify effective neutralizing antibodies for SARS-CoV-2, the binding affinities of neutralizing antibodies with SARS-CoV-2 S protein were predicted and compared by antibody-antigen docking simulation. The result showed that CR3022 neutralizing antibody from human may have higher binding affinity with SARS-CoV-2 S protein than SARS-CoV S protein. We also found that F26G19 and D12 mouse antibodies could bind to SARS-CoV S protein with high affinity. Our findings provide crucial clues towards the development of antigen diagnosis, therapeutic antibody, and the vaccine against SARS-CoV-2.

negative: Whole genome identification of potential G-quadruplexes and analysis of the G-quadruplex binding domain for SARS-CoV-2 The Coronavirus Disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) quickly become a global public health emergency. G-quadruplex, one of the non-canonical secondary structures, has shown potential antiviral values. However, little is known about G-quadruplexes on the emerging SARS-CoV-2. Herein, we characterized the potential G-quadruplexes both in the positive and negative-sense viral stands. The identified potential G-quadruplexes exhibits similar features to the G-quadruplexes detected in the human transcriptome. Within some bat and pangolin related beta coronaviruses, the G-quartets rather than the loops are under heightened selective constraints. We also found that the SUD-like sequence is retained in the SARS-CoV-2 genome, while some other coronaviruses that can infect humans are depleted. Further analysis revealed that the SARS-CoV-2 SUD-like sequence is almost conserved among 16,466 SARS-CoV-2 samples. And the SARS-CoV-2 SUDcore-like dimer displayed similar electrostatic potential pattern to the SUD dimer. Considering the potential value of G-quadruplexes to serve as targets in antiviral strategy, we hope our fundamental research could provide new insights for the SARS-CoV-2 drug discovery.

negative: [What is the origin of SARS-CoV-2?] Every time a pandemic occurs, dozens of theories emerge to attribute the origin of the event to different facts. The COVID-19 pandemic that has hit virtually all the globe has been no exception. What is known so far about the origin of the virus that causes COVID 19? The first investigations on the origin of this disease have determined that it is a new type of virus, the origin of which is most likely zoonotic.

negative: GenomeLandscaper: Landscape analysis of genome-fingerprints maps assessing chromosome architecture Assessing correctness of an assembled chromosome architecture is a central challenge. We create a geometric analysis method (called GenomeLandscaper) to conduct landscape analysis of genome-fingerprints maps (GFM), trace large-scale repetitive regions, and assess their impacts on the global architectures of assembled chromosomes. We develop an alignment-free method for phylogenetics analysis. The human Y chromosomes (GRCh.chrY, HuRef.chrY and YH.chrY) are analysed as a proof-of-concept study. We construct a galaxy of genome-fingerprints maps (GGFM) for them, and a landscape compatibility among relatives is observed. But a long sharp straight line on the GGFM breaks such a landscape compatibility, distinguishing GRCh38p1.chrY (and throughout GRCh38p7.chrY) from GRCh37p13.chrY, HuRef.chrY and YH.chrY. We delete a 1.30-Mbp target segment to rescue the landscape compatibility, matching the antecedent GRCh37p13.chrY. We re-locate it into the modelled centromeric and pericentromeric region of GRCh38p10.chrY, matching a gap placeholder of GRCh37p13.chrY. We decompose it into sub-constituents (such as BACs, interspersed repeats, and tandem repeats) and trace their homologues by phylogenetics analysis. We elucidate that most examined tandem repeats are of reasonable quality, but the BAC-sized repeats, 173U1020C (176.46 Kbp) and 5U41068C (205.34 Kbp), are likely over-repeated. These results offer unique insights into the centromeric and pericentromeric regions of the human Y chromosomes.

negative: Interfacial Water Molecules Make RBD of SPIKE Protein and Human ACE2 to Stick Together A novel coronavirus (SARS-CoV-2; COVID-19) that initially originates from Wuhan province in China has emerged as a global pandemic, an outbreak that started at the end of 2019 which claims 431,192 (Date: 15th June 2020 (https://covid19.who.in) life till now. Since then scientists all over the world are engaged in developing new vaccines, antibodies, or drug molecules to combat this new threat. Here in this work, we performed an in-silico analysis on the protein-protein interactions between the receptor-binding (RBD) domain of viral SPIKE protein and human angiotensin-converting enzyme 2 (hACE2) receptor to highlight the key alteration that happened from SARS-CoV to SARS-CoV-2. We analyzed and compared the molecular differences between these two viruses by using various computational approaches such as binding affinity calculations, computational alanine, and molecular dynamics simulations. The binding affinity calculations show SARS-CoV-2 binds little more firmly to the hACE2 receptor than that of SARS-CoV. Analysis of simulation trajectories reveals that enhanced hydrophobic contacts or the van der Waals interaction play a major role in stabilizing the protein-protein interface. The major finding obtained from molecular dynamics simulations is that the RBD-ACE2 interface is populated with water molecules and interacts strongly with both RBD and ACE2 interfacial residues during the simulation periods. We also emphasize that the interfacial water molecules play a critical role in binding and maintaining the stability of the RBD/hACE2 complex. The water-mediated hydrogen bond by the bridge water molecules is crucial for stabilizing the RBD and ACE2 domains. The structural and dynamical features presented here may serve as a guide for developing new drug molecules, vaccines, or antibodies to combat the COVID-19 pandemic.

negative: Demographic, etiological, and histological pulmonary analysis of patients with acute respiratory failure: a study of 19 years of autopsies INTRODUCTION: Acute respiratory failure has been one of the most important causes of death in intensive care units, and certain aspects of its pulmonary pathology are currently unknown. OBJECTIVES: The objective was to describe the demographic data, etiology, and pulmonary histopathological findings of different diseases in the autopsies of patients with acute respiratory failure. METHOD: Autopsies of 4,710 patients with acute respiratory failure from 1990 to 2008 were reviewed, and the following data were obtained: age, sex, and major associated diseases. The pulmonary histopathology was categorized as diffuse alveolar damage, pulmonary edema, alveolar hemorrhage, and lymphoplasmacytic interstitial pneumonia. The odds ratio of the concordance between the major associated diseases and specific autopsy findings was calculated using logistic regression. RESULTS: Bacterial bronchopneumonia was present in 33.9% of the cases and cancer in 28.1%. The pulmonary histopathology showed diffuse alveolar damage in 40.7% (1,917) of the cases. A multivariate analysis showed a significant and powerful association between diffuse alveolar damage and bronchopneumonia, HIV/AIDS, sepsis, and septic shock, between liver cirrhosis and pulmonary embolism, between pulmonary edema and acute myocardial infarction, between dilated cardiomyopathy and cancer, between alveolar hemorrhage and bronchopneumonia and pulmonary embolism, and between lymphoplasmacytic interstitial pneumonia and HIV/AIDS and liver cirrhosis. CONCLUSIONS: Bronchopneumonia was the most common diagnosis in these cases. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Further studies are necessary to elucidate the complete pathophysiological mechanisms involved with each disease and the development of acute respiratory failure.

negative: Response to “Comments on "Homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross‐species transmission from snake to human” and “Codon bias analysis alone is uninformative for identifying host(s) of new virus” We have recently reported for the first time that SARS‐CoV‐2 maybe a bat‐originated coronavirus with a recombination occurred within the spike (S) protein gene based on phylogenetic and simplot analyses(1). These two conclusions are supported by findings recently reported by others and are well accepted in the field of SARS‐CoV‐2 research(2‐4). This article is protected by copyright. All rights reserved.

negative: SARS-CoV-2 epidemic in India: epidemiological features and in silico analysis of the effect of interventions Background: After SARS-CoV-2 set foot in India, the Government took a number of steps to limit the spread of the virus in the country. This included restricted testing, isolation, contact tracing and quarantine, and enforcement of a nation-wide lockdown starting 25 March 2020. The objectives of this study were to i) describe the age,gender distribution and mortality among COVID-19 patients identified till 14 April 2020 and predict the range of contact rate; and ii) predict the number of active COVID-19 patients after 40 days of lockdown. Methods: We used a cross-sectional descriptive design for first objective and a susceptible-infected-removed model for in silico predictions. We collected data from government-controlled and crowdsourced websites. Results: Studying age and gender parameters of 1161 Indian COVID-19 patients, the median age was 38 years (IQR, 27-52) with 20-39 year-old males being the most affected group. The number of affected patients were 854 (73.6%) men and 307 (26.4%) women. If the current contact rate continues (0.25-27), India may have 110460 to 220575 infected persons at the end of 40 days lockdown. Conclusion: The disease is majorly affecting a younger age group in India. Interventions have been helpful in preventing the worst-case scenario in India, but will be unable to prevent the spike in number of cases.

negative: Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9 Summary Many of the SARS-CoV-2 proteins have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate viral replication, overall virulence and viral genomic RNA reproduction. We sought to better characterise the SARS-CoV-2 Nsp9 and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The SARS-CoV-2 Nsp9 structure revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted the relative juxtapositioning of the monomers within the homodimer. We have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that warrants further study to understanding Nsp9 function.

negative: Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis BACKGROUND: The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. RESULTS: Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. CONCLUSIONS: Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

negative: Master Regulator Analysis of the SARS-CoV-2/Human Interactome The recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. In this paper, we set out to shed light on the SARS-CoV-2/host receptor recognition, a crucial factor for successful virus infection. Based on the current knowledge of the interactome between SARS-CoV-2 and host cell proteins, we performed Master Regulator Analysis to detect which parts of the human interactome are most affected by the infection. We detected, amongst others, affected apoptotic and mitochondrial mechanisms, and a downregulation of the ACE2 protein receptor, notions that can be used to develop specific therapies against this new virus.

negative: Serological Approaches for COVID-19: Epidemiologic Perspective on Surveillance and Control Since December 2019, the novel coronavirus, SARS-CoV-2, has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. Early detection of SARS-CoV-2 is one of the crucial interventions to control virus spread and dissemination. Molecular assays have been the gold standard to directly detect for the presence of viral genetic material in infected individuals. However, insufficient viral RNA at the point of detection may lead to false negative results. As such, it is important to also employ immune-based assays to determine one's exposure to SARS-CoV-2, as well as to assist in the surveillance of individuals with prior exposure to SARS-CoV-2. Within a span of 4 months, extensive studies have been done to develop serological systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during SARS-CoV-2 infection. The vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. This review aims to provide a concise yet extensive collation of current immunoassays for SARS-CoV-2, while discussing the strengths, limitations and applications of antibody detection in SARS-CoV-2 research and control.

negative: Respiratory Viruses Respiratory viruses are the most frequent causative agents of disease in humans, with significant impact on morbidity and mortality worldwide. Common respiratory agents from several virus families are well adapted to efficient person-to-person transmission and circulate in a global scale, and community-based studies conducted over the past five decades or so confirm that these viruses are the predominant etiological agents of acute respiratory infections (ARIs). The respiratory viruses that most commonly circulate in all continents as endemic or epidemic agents are influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses. Although vaccines and effective antiviral drugs are not yet available for most of these viruses, much progress has been made in the understanding of their biology and fundamental issues of host–parasite relationship. This article is a summary of the current knowledge about these viruses.

negative: An updated min-review on environmental route of the SARS-CoV-2 transmission The risk of newly emerging diseases is constantly present in a world where changes occur significantly in climatic, commercial, and ecological conditions, in addition to the development of biomedical investigations in new situations. An epidemic respiratory disease instigated by a new coronavirus was initially identified in and has resulted in the current global dissemination. This viral strain and its related disease has been termed “SARS-CoV-2” and “coronavirus disease 2019” (abbreviated “COVID-19” or “2019-nCoV”), respectively, which is transmitted simply between individuals. The World Health Organization (WHO) announced the COVID-19 outburst as a pandemic on March 11, which necessitates a cooperative endeavour globally for mitigating the spread of COVID-19. The absence of previous, and minimum present-day information, particularly concerning the path of contagion have precluded the control of this disease. The present article, therefore, describes the SARS-CoV-2 paths of contagion such as drinking water, solid waste, sewer water, ambient air, and the rest of emerging likely paths.

negative: Phylodynamic analysis and molecular diversity of the avian infectious bronchitis virus of chickens in Brazil Abstract Avian infectious bronchitis virus (IBV) is the etiological agent of a highly contagious disease, which results in severe economic losses to the poultry industry. The spike protein (S1 subunit) is responsible for the molecular diversity of the virus and many sero/genotypes are described around the world. Recently a new standardized classification of the IBV molecular diversity was conducted, based on phylogenetic analysis of the S1 gene sequences sampled worldwide. Brazil is one of the biggest poultry producers in the world and the present study aimed to review the molecular diversity and reconstruct the evolutionary history of IBV in the country. All IBV S1 gene sequences, with local and year of collection information available on GenBank, were retrieved. Phylogenetic analyses were carried out based on a maximum likelihood method for the classification of genotypes occurring in Brazil, according to the new classification. Bayesian phylogenetic analyses were performed with the Brazilian clade and related international sequences to determine the evolutionary history of IBV in Brazil. A total of 143 Brazilian sequences were classified as GI-11 and 46 as GI-1 (Mass). Within the GI-11 clade, we have identified a potential recombinant strain circulating in Brazil. Phylodynamic analysis demonstrated that IBV GI-11 lineage was introduced in Brazil in the 1950s (1951, 1917–1975 95% HPD) and population dynamics was mostly constant throughout the time. Despite the national vaccination protocols, our results show the widespread dissemination and maintenance of the IBV GI-11 lineage in Brazil and highlight the importance of continuous surveillance to evaluate the impact of currently used vaccine strains on the observed viral diversity of the country.

negative: Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.

negative: Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97 7% The homology of M gene was the highest (99 0%) However, the homology of ORF3 gene was 97 7%, and protein of ORF3 was 90 1% In addition, HLJBY showed the highest nucleotide identity (99 9%) with PEDV-SX/China/2017 strain and lowest similarity (91 2%) to PEDV/Belgorod/dom/2008 strain We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains The highest nucleotide identity was 99 9% compared with PEDV-SX/China/2017 strain, and the lowest nucleotide identity was 60 0% compared with PEDV/Belgorod/dom/2008 strain The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa) Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59-61, 144) Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80-224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71 4%-98 9% ORF3 protein of both HLJBY strain and PEDV-SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY-P10 or PEDV CV777-P8 inoculated piglets

negative: Docetaxel induces moderate ovarian toxicity in mice, primarily affecting granulosa cells of early growing follicles Advances in cancer therapy have focused attention on the quality of life of cancer survivors. Since infertility is a major concern following chemotherapy, it is important to characterize the drug-specific damage to the reproductive system to help find appropriate protective strategies. This study investigates the damage on neonatal mouse ovary maintained in vitro for 6 days, and exposed for 24 h (on Day 2) to clinically relevant doses of Docetaxel (DOC; low: 0.1 µM, mid: 1 µM, high: 10 µM). Furthermore, the study explores the putative protective action exerted by Tri-iodothyronine (T3; 10(−7) M). At the end of culture, morphological analyses and follicle counts showed that DOC negatively impacts on early growing follicles, decreasing primary follicle number and severely affecting health at the transitional and primary stages. Poor follicle health was mainly due to effects on granulosa cells, indicating that the effects of DOC on oocytes were likely to be secondary to granulosa cell damage. DOC damages growing follicles specifically, with no direct effect on the primordial follicle reserve. Immunostaining and western blotting showed that DOC induces activation of intrinsic, type II apoptosis in ovarian somatic cells; increasing the levels of cleaved caspase 3, cleaved caspase 8, Bax and cleaved poly(ADP-ribose) polymerase, while also inducing movement of cytochrome C from mitochondria into the cytosol. T3 did not prevent the damage induced by the low dose of DOC. These results demonstrated that DOC induces a gonadotoxic effect on the mouse ovary through induction of somatic cell apoptosis, with no evidence of direct effects on the oocyte, and that the damaging effect is not mitigated by T3.

negative: The First 75 Days of Novel Coronavirus (SARS-CoV-2) Outbreak: Recent Advances, Prevention, and Treatment The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously known as 2019-nCoV) outbreak has engulfed an unprepared world amidst a festive season. The zoonotic SARS-CoV-2, believed to have originated from infected bats, is the seventh member of enveloped RNA coronavirus. Specifically, the overall genome sequence of the SARS-CoV-2 is 96.2% identical to that of bat coronavirus termed BatCoV RaTG13. Although the current mortality rate of 2% is significantly lower than that of SARS (9.6%) and Middle East respiratory syndrome (MERS) (35%), SARS-CoV-2 is highly contagious and transmissible from human to human with an incubation period of up to 24 days. Some statistical studies have shown that, on average, one infected patient may lead to a subsequent 5.7 confirmed cases. Since the first reported case of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 on December 1, 2019, in Wuhan, China, there has been a total of 60,412 confirmed cases with 1370 fatalities reported in 25 different countries as of February 13, 2020. The outbreak has led to severe impacts on social health and the economy at various levels. This paper is a review of the significant, continuous global effort that was made to respond to the outbreak in the first 75 days. Although no vaccines have been discovered yet, a series of containment measures have been implemented by various governments, especially in China, in the effort to prevent further outbreak, whilst various medical treatment approaches have been used to successfully treat infected patients. On the basis of current studies, it would appear that the combined antiviral treatment has shown the highest success rate. This review aims to critically summarize the most recent advances in understanding the coronavirus, as well as the strategies in prevention and treatment.

negative: Structure, Immunopathogenesis and Vaccines Against SARS Coronavirus A new disease, severe atypical respiratory syndrome (SARS), emerged in China in late 2002 and developed into the first epidemic of the 21st century. The disease was caused by an unknown animal coronavirus (CoV) that had crossed the species barrier through close contact of humans with infected animals, and was identified as the etiological agent for SARS. This new CoV not only became readily transmissible between humans but also was also more pathogenic. The disease spread across the world rapidly due to the air travel, and infected 8096 people and caused 774 deaths in 26 countries on 5 continents. The disease is characterized by flu-like symptoms, including high fever, malaise, cough, diarrhea, and infiltrates visible on chest radiography. The overall mortality was about 10%, but varied profoundly with age; the course of disease seemed to be milder in the pediatric age group and resulted rarely in a fatal outcome, but the mortality in the elderly was as high as 50%. Aggressive quarantine measures taken by the health authorities have successfully contained and terminated the disease transmission. As a result there are no SARS cases recorded recently. Nevertheless there is a possibility that the disease may emerge in the population with high vigor. Significant progress has been made in understanding the disease biology, pathogenesis, development of animal models, and design and evaluation of different vaccines, and these are the focus of this chapter.

negative: Who is most likely to be infected with SARS-CoV-2?

negative: Use of phylogenetics in the molecular epidemiology and evolutionary studies of viral infections. Since DNA sequencing techniques first became available almost 30 years ago, the amount of nucleic acid sequence data has increased enormously. Phylogenetics, which is widely applied to compare and analyze such data, is particularly useful for the analysis of genes from rapidly evolving viruses. It has been used extensively to describe the molecular epidemiology and transmission of the human immunodeficiency virus (HIV), the origins and subsequent evolution of the severe acute respiratory syndrome (SARS)-associated coronavirus (SCoV), and, more recently, the evolving epidemiology of avian influenza as well as seasonal and pandemic human influenza viruses. Recent advances in phylogenetic methods can infer more in-depth information about the patterns of virus emergence, adding to the conventional approaches in viral epidemiology. Examples of this information include estimations (with confidence limits) of the actual time of the origin of a new viral strain or its emergence in a new species, viral recombination and reassortment events, the rate of population size change in a viral epidemic, and how the virus spreads and evolves within a specific population and geographical region. Such sequence-derived information obtained from the phylogenetic tree can assist in the design and implementation of public health and therapeutic interventions. However, application of many of these advanced phylogenetic methods are currently limited to specialized phylogeneticists and statisticians, mainly because of their mathematical basis and their dependence on the use of a large number of computer programs. This review attempts to bridge this gap by presenting conceptual, technical, and practical aspects of applying phylogenetic methods in studies of influenza, HIV, and SCoV. It aims to provide, with minimal mathematics and statistics, a practical overview of how phylogenetic methods can be incorporated into virological studies by clinical and laboratory specialists.

negative: Wild animal surveillance for coronavirus HKU1 and potential variants of other coronaviruses. 1. Although CoV-HKU1 was not identified in any of the studied animals, a coronavirus closely related to SARS-CoV (bat-SARS-CoV) was identified in 23 (19%) of 118 wild Chinese horseshoe bats by reverse transcriptase polymerase chain reaction (RT-PCR). 2. Complete genome sequencing and phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b coronaviruses, distantly related to known group 2 coronaviruses. 3. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike gene. The presence of a29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. 4. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats using an enzyme immunoassay.Neutralising antibody to human SARS-CoV was also detected in those with lower viral loads.5. This study also revealed a previously unknown diversity of coronaviruses in bats, which are important natural reservoir for coronaviruses including SARS-CoV-like viruses.

negative: Cloning and Sequence Analysis of N Gene of Transmissible Gastroenteritis Virus HYM-09 Isolated from Dog in China Transmissible gastroenteritis virus (TGEV) is the etiological agent of TGE, and dogs are potential carriers of TGEV. In this study, genomic RNA were extracted from TGEV designated HYM-09 isolated from dog naturally infected with TGEV. The nucleocapsid (N) gene of HYM-09 was amplified by RT-PCR and cloned into pMD18-T vector. The N gene cDNA was sequenced and encompassed an open reading frame of 1,149 nucleotides, encoding a 382-amino acids protein. Sequence analyses of the N genes were performed, including homologous comparison, phylogenetic tree analysis and residue substitution analysis. The results showed that there existed some unique mutations in the HYM-09 isolate N gene, but HYM-09 N gene shared over 96 % homologous identities compared with 12 TGEV reference strains derived from other regions or countries respectively. The phylogenetic tree analysis revealed that the HYM-09 branched into the most strains group. This study shows that the nucleotide sequence analysis can form a base or further study on the mutation trend of non-porcine TGEV.

negative: A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing There is currently an urgent unmet need to increase coronavirus disease 2019 (COVID-19) testing capability to effectively respond to the COVID-19 pandemic. However, the current shortage in RNA extraction reagents as well as limitations in qPCR protocols have resulted in bottlenecks in testing capacity. Herein, we describe a novel molecular diagnostic for COVID-19 based on Sanger sequencing. This assay uses the addition of a frame-shifted spike-in, a modified PCR master mix, and custom Sanger sequencing data analysis to detect and quantify SARS-CoV-2 RNA at a limit of detection comparable to existing qPCR-based assays, at 10-20 genome copy equivalents. Crucially, our assay was able to detect SARS-CoV-2 RNA from viral particles suspended in transport media that was directly added to the PCR master mix, suggesting that RNA extraction can be skipped entirely without any degradation of test performance. Since Sanger sequencing instruments are widespread in clinical laboratories and commonly have built-in liquid handling automation to support up to 3840 samples per instrument per day, the widespread adoption of qSanger COVID-19 diagnostics can unlock more than 1,000,000 tests per day in the US.

negative: Advances in disease diagnosis, vaccine development and other emerging methods to control pathogens in aquaculture Disease is still regarded as a major constraint to aquaculture production globally. Rapid disease diagnosis and vaccination play a huge part in the control of bacterial diseases, and there has been significant progress in both of these areas. This chapter considers the limitations of existing methods and reviews recent advances made in pathogen detection technologies and vaccine development methodologies. Future directions are discussed, including nanotechnology and reversed vaccinology.

negative: Phylogenetic and recombination analysis of coronavirus HKU1, a novel coronavirus from patients with pneumonia Phylogenetic trees constructed using predicted amino acid sequences of putative proteins of coronavirus HKU1 (CoV-HKU1) revealed that CoV-HKU1 formed a distinct branch among group 2 coronaviruses. Of the 14 trees from p65 to nsp10, nine showed that CoV-HKU1 was clustered with murine hepatitis virus. From nsp11, the topologies of the trees changed dramatically. For the eight trees from nsp11 to N, seven showed that the CoV-HKU1 branch was the first branch. The codon usage patterns of CoV-HKU1 differed significantly from those in other group 2 coronaviruses. Split decomposition analysis revealed that recombination events had occurred between CoV-HKU1 and other coronaviruses.

negative: Evidence of Increasing Diversification of Emerging SARS‐CoV‐2 Strains BACKGROUND: On January 30th, 2020, an outbreak of atypical pneumonia caused by a novel Betacoronavirus (βCoV), named SARS‐CoV‐2, was declared a public health emergency of international concern by the World Health Organization. For this reason, a detailed evolutionary analysis of SARS‐CoV‐2 strains currently circulating in different geographic regions of the world was performed. METHODS: A compositional analysis as well as a Bayesian coalescent analysis of complete genome sequences of SARS‐CoV‐2 strains recently isolated in Europe, North America, South America and Asia was performed. RESULTS: The results of these studies revealed a diversification of SARS‐CoV‐2 strains in three different genetic clades. Co‐circulation of different clades in different countries, as well as different genetic lineages within different clades were observed. The time of the most recent common ancestor (tMRCA) was established to be around November 1, 2019. A mean rate of evolution of 6.57 x 10(‐4) substitutions per site per year was found. A significant migration rate per genetic lineage per year from Europe to South America was also observed. CONCLUSION: The results of these studies revealed an increasing diversification of SARS‐CoV‐2 strains. High evolutionary rates and fast population growth characterizes the population dynamics of SARS‐CoV‐2 strains. This article is protected by copyright. All rights reserved.

negative: periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data We have developed periscope, a tool for the detection and quantification of sub-genomic RNA in ARTIC network protocol generated Nanopore SARS-CoV-2 sequence data. We applied periscope to 1155 SARS-CoV-2 sequences from Sheffield, UK. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sub-genomic RNA. We were able to detect all canonical sub-genomic RNAs at expected abundances, with the exception of ORF10, suggesting that this is not a functional ORF. A number of recurrent non-canonical sub-genomic RNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sub-genomic RNA analysis. Finally variants found in genomic RNA are transmitted to sub-genomic RNAs with high fidelity in most cases. This tool can be applied to tens of thousands of sequences worldwide to provide the most comprehensive analysis of SARS-CoV-2 sub-genomic RNA to date.

negative: Letea Virus: Comparative Genomics and Phylogenetic Analysis of a Novel Reassortant Orbivirus Discovered in Grass Snakes (Natrix natrix) The discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. Arbovirus surveillance is not conducted regularly in Romania, being particularly very scarce in the remote and diverse areas like the Danube Delta. Here we describe the detection and genetic characterization of a novel orbivirus (Reoviridae: Orbivirus) designated as Letea virus, which was found in grass snakes (Natrix natrix) during a metagenomic and metatranscriptomic survey conducted between 2014 and 2017. This virus is the first orbivirus discovered in reptiles. Phylogenetic analyses placed Letea virus as a highly divergent species in the Culicoides-/sand fly-borne orbivirus clade. Gene reassortment and intragenic recombination were detected in the majority of the nine Letea virus strains obtained, implying that these mechanisms play important roles in the evolution and diversification of the virus. However, the screening of arthropods, including Culicoides biting midges collected within the same surveillance program, tested negative for Letea virus infection and could not confirm the arthropod vector of the virus. The study provided complete genome sequences for nine Letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. The phylogenetic associations warrant further screening of arthropods, as well as sustained surveillance efforts for elucidation of Letea virus natural cycle and possible implications for animal and human health.

negative: Broad and differential animal ACE2 receptor usage by SARS-CoV-2 The COVID-19 pandemic has caused an unprecedented global public health and economy crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2 from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activity, respectively. Among the remaining species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.

negative: An Exploratory Characterization of Bugs in COVID-19 Software Projects Context: The dire consequences of the COVID-19 pandemic has influenced development of COVID-19 software i.e., software used for analysis and mitigation of COVID-19. Bugs in COVID-19 software can be consequential, as COVID-19 software projects can impact public health policy and user data privacy. Objective: The goal of this paper is to help practitioners and researchers improve the quality of COVID-19 software through an empirical study of open source software projects related to COVID-19. Methodology: We use 129 open source COVID-19 software projects hosted on GitHub to conduct our empirical study. Next, we apply qualitative analysis on 550 bug reports from the collected projects to identify bug categories. Findings: We identify 8 bug categories, which include data bugs i.e., bugs that occur during mining and storage of COVID-19 data. The identified bug categories appear for 7 categories of software projects including (i) projects that use statistical modeling to perform predictions related to COVID-19, and (ii) medical equipment software that are used to design and implement medical equipment, such as ventilators. Conclusion: Based on our findings, we advocate for robust statistical model construction through better synergies between data science practitioners and public health experts. Existence of security bugs in user tracking software necessitates development of tools that will detect data privacy violations and security weaknesses.

negative: A Brief Review: The Z-curve Theory and its Application in Genome Analysis In theoretical physics, there exist two basic mathematical approaches, algebraic and geometrical methods, which, in most cases, are complementary. In the area of genome sequence analysis, however, algebraic approaches have been widely used, while geometrical approaches have been less explored for a long time. The Z-curve theory is a geometrical approach to genome analysis. The Z-curve is a three-dimensional curve that represents a given DNA sequence in the sense that each can be uniquely reconstructed given the other. The Z-curve, therefore, contains all the information that the corresponding DNA sequence carries. The analysis of a DNA sequence can then be performed through studying the corresponding Z-curve. The Z-curve method has found applications in a wide range of areas in the past two decades, including the identifications of protein-coding genes, replication origins, horizontally-transferred genomic islands, promoters, translational start sides and isochores, as well as studies on phylogenetics, genome visualization and comparative genomics. Here, we review the progress of Z-curve studies from aspects of both theory and applications in genome analysis.

negative: A novel bat coronavirus closely related to SARS-CoV-2 contains natural insertions at the S1/S2 cleavage site of the spike protein Summary The unprecedented pandemic of pneumonia caused by a novel coronavirus, SARS-CoV-2, in China and beyond has had major public health impacts on a global scale [1,2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats collected from Yunnan Province in China between May and October, 2019. Notably, RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity (61.3%) to SARS-CoV-2 in the receptor binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong evidence that such insertion events can occur naturally in animal betacoronaviruses.

negative: Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene Abstract Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.

negative: ZCURVE_CoV: a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing SARS-CoV genomes Abstract A new system to recognize protein coding genes in the coronavirus genomes, specially suitable for the SARS-CoV genomes, has been proposed in this paper. Compared with some existing systems, the new program package has the merits of simplicity, high accuracy, reliability, and quickness. The system ZCURVE_CoV has been run for each of the 11 newly sequenced SARS-CoV genomes. Consequently, six genomes not annotated previously have been annotated, and some problems of previous annotations in the remaining five genomes have been pointed out and discussed. In addition to the polyprotein chain ORFs 1a and 1b and the four genes coding for the major structural proteins, spike (S), small envelop (E), membrane (M), and nuleocaspid (N), respectively, ZCURVE_CoV also predicts 5–6 putative proteins in length between 39 and 274 amino acids with unknown functions. Some single nucleotide mutations within these putative coding sequences have been detected and their biological implications are discussed. A web service is provided, by which a user can obtain the annotated result immediately by pasting the SARS-CoV genome sequences into the input window on the web site (http://tubic.tju.edu.cn/sars/). The software ZCURVE_CoV can also be downloaded freely from the web address mentioned above and run in computers under the platforms of Windows or Linux.

negative: What's Old is New! Similarities Between SARS-CoV-2 and HIV

negative: A novel feature-based method for whole genome phylogenetic analysis without alignment: application to HEV genotyping and subtyping. Traditional phylogenetic analysis is based on multiple sequence alignment. With the development of worldwide genome sequencing project, more and more completely sequenced genomes become available. However, traditional sequence alignment tools are impossible to deal with large-scale genome sequence. So, the development of new algorithms to infer phylogenetic relationship without alignment from whole genome information represents a new direction of phylogenetic study in the post-genome era. In the present study, a novel algorithm based on BBC (base-base correlation) is proposed to analyze the phylogenetic relationships of HEV (Hepatitis E virus). When 48 HEV genome sequences are analyzed, the phylogenetic tree that is constructed based on BBC algorithm is well consistent with that of previous study. When compared with methods of sequence alignment, the merit of BBC algorithm appears to be more rapid in calculating evolutionary distances of whole genome sequence and not requires any human intervention, such as gene identification, parameter selection. BBC algorithm can serve as an alternative to rapidly construct phylogenetic trees and infer evolutionary relationships.

negative: A novel model for protein sequence similarity analysis based on spectral radius Abstract Advances in sequencing technologies led to rapid increase in the number and diversity of biological sequences, which facilitated development in the sequence research. In this paper, we present a new method for analyzing protein sequence similarity. We calculated the spectral radii of 20 amino acids (AAs) and put forward a novel 2-D graphical representation of protein sequences. To characterize protein sequences numerically, three groups of features were extracted and related to statistical, dynamics measurements and fluctuation complexity of the sequences. With the obtained feature vector, two models utilizing Gaussian Kernel similarity and Cosine similarity were built to measure the similarity between sequences. We applied our method to analyze the similarities/dissimilarities of four data sets. Both proposed models received consistent results with improvements when compared to that obtained by the ClustalW analysis. The novel approach we present in this study may therefore benefit protein research in medical and scientific fields.

negative: State-of-the-art tools to identify druggable protein ligand of SARS-CoV-2 INTRODUCTION: The SARS-CoV-2 (previously 2019-nCoV) outbreak in Wuhan, China and other parts of the world affects people and spreads coronavirus disease 2019 (COVID-19) through human-to-human contact, with a mortality rate of > 2%. There are no approved drugs or vaccines yet available against SARS-CoV-2. MATERIAL AND METHODS: State-of-the-art tools based on in-silico methods are a cost-effective initial approach for identifying appropriate ligands against SARS-CoV-2. The present study developed the 3D structure of the envelope and nucleocapsid phosphoprotein of SARS-CoV-2, and molecular docking analysis was done against various ligands. RESULTS: The highest log octanol/water partition coefficient, high number of hydrogen bond donors and acceptors, lowest non-bonded interaction energy between the receptor and the ligand, and high binding affinity were considered for the best ligand for the envelope (mycophenolic acid: log P = 3.00; DG = –10.2567 kcal/mol; pKi = 7.713 µM) and nucleocapsid phosphoprotein (1-[(2,4-dichlorophenyl)methyl]pyrazole-3,5-dicarboxylic acid: log P = 2.901; DG = –12.2112 kcal/mol; pKi = 7.885 µM) of SARS-CoV-2. CONCLUSIONS: The study identifies the most potent compounds against the SARS-CoV-2 envelope and nucleocapsid phosphoprotein through state-of-the-art tools based on an in-silico approach. A combination of these two ligands could be the best option to consider for further detailed studies to develop a drug for treating patients infected with SARS-CoV-2, COVID-19.

negative: Virus Pathogen Database and Analysis Resource (ViPR): A Comprehensive Bioinformatics Database and Analysis Resource for the Coronavirus Research Community Several viruses within the Coronaviridae family have been categorized as either emerging or re-emerging human pathogens, with Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) being the most well known. The NIAID-sponsored Virus Pathogen Database and Analysis Resource (ViPR, www.viprbrc.org) supports bioinformatics workflows for a broad range of human virus pathogens and other related viruses, including the entire Coronaviridae family. ViPR provides access to sequence records, gene and protein annotations, immune epitopes, 3D structures, host factor data, and other data types through an intuitive web-based search interface. Records returned from these queries can then be subjected to web-based analyses including: multiple sequence alignment, phylogenetic inference, sequence variation determination, BLAST comparison, and metadata-driven comparative genomics statistical analysis. Additional tools exist to display multiple sequence alignments, view phylogenetic trees, visualize 3D protein structures, transfer existing reference genome annotations to new genomes, and store or share results from any search or analysis within personal private ‘Workbench’ spaces for future access. All of the data and integrated analysis and visualization tools in ViPR are made available without charge as a service to the Coronaviridae research community to facilitate the research and development of diagnostics, prophylactics, vaccines and therapeutics against these human pathogens.

negative: Genomic Characterization and Phylogenetic Classification of Bovine Coronaviruses Through Whole Genome Sequence Analysis Bovine coronavirus (BCoV) is zoonotically transmissible among species, since BCoV-like viruses have been detected in wild ruminants and humans. BCoV causing enteric and respiratory disease is widespread in cattle farms worldwide; however, limited information is available regarding the molecular characterization of BCoV because of its large genome size, despite its significant economic impact. This study aimed to better understand the genomic characterization and evolutionary dynamics of BCoV via comparative sequence and phylogenetic analyses through whole genome sequence analysis using 67 BCoV isolates collected throughout Japan from 2006 to 2017. On comparing the genomic sequences of the 67 BCoVs, genetic variations were detected in 5 of 10 open reading frames (ORFs) in the BCoV genome. Phylogenetic analysis using whole genomes from the 67 Japanese BCoV isolates in addition to those from 16 reference BCoV strains, revealed the existence of two major genotypes (classical and US wild ruminant genotypes). All Japanese BCoV isolates originated from the US wild ruminant genotype, and they tended to form the same clusters based on the year and farm of collection, not the disease type. Phylogenetic trees on hemagglutinin-esterase protein (HE), spike glycoprotein (S), nucleocapsid protein (N) genes and ORF1 revealed clusters similar to that on whole genome, suggesting that the evolution of BCoVs may be closely associated with variations in these genes. Furthermore, phylogenetic analysis of BCoV S genes including those of European and Asian BCoVs and human enteric coronavirus along with the Japanese BCoVs revealed that BCoVs differentiated into two major types (European and American types). Moreover, the European and American types were divided into eleven and three genotypes, respectively. Our analysis also demonstrated that BCoVs with different genotypes periodically emerged and predominantly circulated within the country. These findings provide useful information to elucidate the detailed molecular characterization of BCoVs, which have spread worldwide. Further genomic analyses of BCoV are essential to deepen the understanding of the evolution of this virus.

negative: Complete genome characterization and phylogenetic analysis of WU polyomavirus in Thai pediatric patients with respiratory tract infections in 2013. BACKGROUND The WU polyomavirus (WUPyV) is a small DNA virus (family Polyomaviridae) that contains a circular double-stranded DNA genome, approximately 5 kb in length. WUPyV was first discovered in the respiratory tract of children with acute respiratory symptoms. OBJECTIVE This study focuses on the complete genome characterization and phylogenetic analysis of WUPyV obtained from Thai patients with respiratory diseases in 2013. MATERIAL AND METHOD DNA was extracted from nasopharyngeal (NP) suction specimens (n = 614) from patients with respiratory tract infections. WUPyV was detected by using semi-nested PCR and then characterized by whole genome sequencing. The nucleotides and deduced amino acid sequences were analyzed by multiple sequences alignment and phylogenetic tree. RESULTS Analysis revealed that 0.16% (1/614) of the sample was positive for WUPyV. Phylogenetic trees demonstrated that WUPyV(isolate CU_Chonburi 3) was closely related to previously described WUPyV. Moreover, whole genome sequences alignment of WUPyV showed several nucleotide variations within non-coding regions and amino acid changes in VP1 (position S347T); VP2 (positions L40V, G120R, Y121I, P123R, G127S, L137F, Q287R, and A327V); LTAg (positions Q357P, V369E, E377K, D378V, A381T, R382E, R383G, and D389G); and, STAg (positions R139S, K141E, R148K, and W153C). CONCLUSION Nucleotide variations within non-coding regions and critical amino acid substitutions in viral proteins may affect the rate of viral replication and viral adaptation; factors that may be linked to the susceptibility and severity of viral infection. Data obtained from this study may be useful in better understanding the genetic characterization and mutation patterns of WUPyV.

negative: Current development of COVID-19 diagnostics, vaccines and therapeutics Abstract A novel coronavirus, designated as SARS-CoV-2, first emerged in Wuhan City, Hubei Province, China, in late December 2019. The rapidly increasing number of cases has caused worldwide panic. In this review, we describe some currently applied diagnostic approaches, as well as therapeutics and vaccines, to prevent, treat and control further outbreaks of SARS-CoV-2 infection.

negative: The Genome sequence of the SARS-associated coronavirus. We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.

negative: SARS-CoV-2 Genome Analysis of Japanese Travelers in Nile River Cruise Japan has reported 26 cases of coronavirus disease 2019 (COVID-19) linked to cruise tours on the River Nile in Egypt between March 5 and 15, 2020. Here, we characterized the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome of isolates from 10 travelers who returned from Egypt and from patients possibly associated with these travelers. We performed haplotype network analysis of SARS-CoV-2 isolates using genome-wide single-nucleotide variations. Our analysis identified two potential Egypt-related clusters from these imported cases, and these clusters were related to globally detected viruses in different countries.

negative: Moderate mutation rate in the SARS coronavirus genome and its implications BACKGROUND: The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) and multiple genomic sequences have been revealed since mid-April, 2003. After a quiet summer and fall in 2003, the newly emerged SARS cases in Asia, particularly the latest cases in China, are reinforcing a wide-spread belief that the SARS epidemic would strike back. With the understanding that SARS-CoV might be with humans for years to come, knowledge of the evolutionary mechanism of the SARS-CoV, including its mutation rate and emergence time, is fundamental to battle this deadly pathogen. To date, the speed at which the deadly virus evolved in nature and the elapsed time before it was transmitted to humans remains poorly understood. RESULTS: Sixteen complete genomic sequences with available clinical histories during the SARS outbreak were analyzed. After careful examination of multiple-sequence alignment, 114 single nucleotide variations were identified. To minimize the effects of sequencing errors and additional mutations during the cell culture, three strategies were applied to estimate the mutation rate by 1) using the closely related sequences as background controls; 2) adjusting the divergence time for cell culture; or 3) using the common variants only. The mutation rate in the SARS-CoV genome was estimated to be 0.80 – 2.38 × 10(-3 )nucleotide substitution per site per year which is in the same order of magnitude as other RNA viruses. The non-synonymous and synonymous substitution rates were estimated to be 1.16 – 3.30 × 10(-3 )and 1.67 – 4.67 × 10(-3 )per site per year, respectively. The most recent common ancestor of the 16 sequences was inferred to be present as early as the spring of 2002. CONCLUSIONS: The estimated mutation rates in the SARS-CoV using multiple strategies were not unusual among coronaviruses and moderate compared to those in other RNA viruses. All estimates of mutation rates led to the inference that the SARS-CoV could have been with humans in the spring of 2002 without causing a severe epidemic.

negative: Endothelial Cells Support Persistent Gammaherpesvirus 68 Infection A variety of human diseases are associated with gammaherpesviruses, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infections usually result in either a productive lytic infection, characterized by expression of all viral genes and rapid cell lysis, or latent infection, characterized by limited viral gene expression and no cell lysis. Here, we report characterization of endothelial cell infection with murine gammaherpesvirus 68 (γHV68), a virus phylogenetically related and biologically similar to the human gammaherpesviruses. Endothelial cells supported γHV68 replication in vitro, but were unique in that a significant proportion of the cells escaped lysis, proliferated, and remained viable in culture for an extended time. Upon infection, endothelial cells became non-adherent and altered in size, complexity, and cell-surface protein expression. These cells were uniformly infected and expressed the lytic transcription program based on detection of abundant viral gene transcripts, GFP fluorescence from the viral genome, and viral surface protein expression. Additionally, endothelial cells continued to produce new infectious virions as late as 30 days post-infection. The outcome of this long-term infection was promoted by the γHV68 v-cyclin, because in the absence of the v-cyclin, viability was significantly reduced following infection. Importantly, infected primary endothelial cells also demonstrated increased viability relative to infected primary fibroblasts, and this increased viability was dependent on the v-cyclin. Finally, we provide evidence for infection of endothelial cells in vivo in immune-deficient mice. The extended viability and virus production of infected endothelial cells indicated that endothelial cells provided a source of prolonged virus production and identify a cell-type specific adaptation of gammaherpesvirus replication. While infected endothelial cells would likely be cleared in a healthy individual, persistently infected endothelial cells could provide a source of continued virus replication in immune-compromised individuals, a context in which gammaherpesvirus-associated pathology frequently occurs.

negative: Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.

negative: Knowledge-based repositioning of the anti-HCV direct antiviral agent Sofosbuvir as SARS-CoV-2 treatment The new human coronavirus named SARS-CoV-2 is a positive-sense RNA virus for which no specific drugs are currently available. A knowledge-based analysis strongly suggests a possible repositioning of the anti-HCV direct antiviral agent (DAA) Sofosbuvir as treatment for SARS-CoV-2. Indeed, the RNA-dependent RNA-polymerases (RdRp) of the two viruses show high sequence and structural homology, supporting the likelihood of binding the Sofosbuvir molecule with similar efficiency. Such a repositioning would allow the containment of the SARS-CoV-2 pandemic and limit the progression of disease to potentially deadly COVID19.

negative: Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8 T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demon-strated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

negative: Molecular epidemiology and phylogeny of Nipah virus infection: A mini review Abstract Nipah virus (NiV) is a member of the genus Henipavirus of the family Paramyxoviridae, characterized by high pathogenicity and endemic in South Asia. It is classified as a Biosafety Level-4 (BSL-4) agent. The case-fatality varies from 40% to 70% depending on the severity of the disease and on the availability of adequate healthcare facilities. At present no antiviral drugs are available for NiV disease and the treatment is just supportive. Phylogenetic and evolutionary analyses can be used to help in understanding the epidemiology and the temporal origin of this virus. This review provides an overview of evolutionary studies performed on Nipah viruses circulating in different countries. Thirty phylogenetic studies have been published from 2000 to 2015 years, searching on pub-med using the key words ‘Nipah virus AND phylogeny’ and twenty-eight molecular epidemiological studies from 2006 to 2015 have been performed, typing the key words ‘Nipah virus AND molecular epidemiology’. Overall data from the published study demonstrated as phylogenetic and evolutionary analysis represent promising tools to evidence NiV epidemics, to study their origin and evolution and finally to act with effective preventive measure.

negative: Isolation of Virus from a SARS Patient and Genome-wide Analysis of Genetic Mutations Related to Pathogenesis and Epidemiology from 47 SARS-CoV Isolates Severe acute respiratory syndrome (SARS) caused by SARS-associated coronavirus (SARS-CoV) is a fatal disease. Prevention of future outbreaks is essential and requires understanding pathogenesis and evolution of the virus. We have isolated a SARS-CoV in China and analyzed 47 SARS-CoV genomes with the aims to reveal the evolution trends of the virus and provide insights into understanding pathogenesis and SARS epidemic. Specimen from a SARS patient was inoculated into cell culture. The presence of SARS-CoV was determined by RT-PCR and confirmed by electron microscopy. Virus was isolated followed by the determination of its genome sequences, which were then analyzed by comparing with other 46 SARS-CoV genomes. Genetic mutations with potential implications to pathogenesis and the epidemic were characterized. This viral genome consists of 29,728 nucleotides with overall organization in agreement with that of published isolates. A total of 348 positions were mutated on 47 viral genomes. Among them 22 had mutations in more than three genomes. Hot spots of nucleotide variations and unique trends of mutations were identified on the viral genomes. Mutation rates were different from gene to gene and were correlated well with periodical or geographic characteristics of the epidemic.

negative: A virus that has gone viral: Amino acid mutation in S protein of Indian isolate of Coronavirus COVID-19 might impact receptor binding and thus infectivity Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019. Spike(S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the 2019 novel coronavirus takes its course throughout the world, more and more sequence analyses are been done and genome sequences getting deposited in various databases. From India two clinical isolates have been sequenced and the full genome deposited in GenBank. We have performed sequence analyses of the spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates one was found to harbour a mutation in its Receptor binding domain (RBD) at position 407. At this site arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-beta branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor ding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.

negative: Initial success in the identification and management of the coronavirus disease 2019 (COVID-19) indicates human-to-human transmission in Wuhan, China Coronavirus (CoV) has been one of the major pandemic threats to human health in the last two decades. The human coronavirus was first identified in 1960s. CoVs 229E, NL63, OC43, HKU1, SARS-CoV, and MERS-CoV have caused numerous disasters or human deaths worldwide. Recently, an outbreak of the previously unknown deadly CoV disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome CoV 2 (SARS-CoV-2, early named 2019-nCoV) occurred in Wuhan, China, and it had caused 81238 cases of confirmed infection, including 3250 deaths until March 19, 2020. Its risks and pandemic potential have brought global consideration. We summarized epidemiology, virological characteristics, clinical symptoms, diagnostic methods, clinical treatments, and prevention methods for COVID-19 to present a reference for the future wave of probable CoV outbreaks.

negative: An analysis of the feasibility of short read sequencing Several methods for ultra high-throughput DNA sequencing are currently under investigation. Many of these methods yield very short blocks of sequence information (reads). Here we report on an analysis showing the level of genome sequencing possible as a function of read length. It is shown that re-sequencing and de novo sequencing of the majority of a bacterial genome is possible with read lengths of 20–30 nt, and that reads of 50 nt can provide reconstructed contigs (a contiguous fragment of sequence data) of 1000 nt and greater that cover 80% of human chromosome 1.

negative: The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19 The devastating effects of the recent global pandemic (termed COVID-19 for “coronavirus disease 2019”) caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10930-020-09901-4) contains supplementary material, which is available to authorized users.

negative: Binding of SARS-CoV-2 and angiotensin-converting enzyme 2: clinical implications

negative: Prevalence, phylogenetic and evolutionary analysis of porcine deltacoronavirus in Henan province, China Abstract Porcine deltacoronavirus (PDCoV) is a novel porcine enteric coronavirus that causes diarrhea, vomiting and dehydration in piglets. This newly virus has spread rapidly and has caused serious economic losses for pig industry since the outbreak in USA in 2014. In this study, 430 faecal and intestinal samples (143 faecal samples and 287 intestinal samples) were collected from individual pigs with diarrhea and 211 serum samples were also collected from the sows with mild diarrhea in 17 regions in Henan province, China from April 2015 to March 2018. The RT-PCR detection indicated that the infection of PDCoV was high up to 23.49% (101/430), and co-infection with PEDV were common (60.40%, 61/101) in Henan pigs. The prevalence of PDCoV in suckling piglets was the highest (36.43%, 94/258). We also found that PDCoV could be detected in sows faeces and sera while the sows showed mild, self-limited diarrhea in clinic. The complete genomes of 4 PDCoV Henan strains (CH-01, HNZK-02, HNZK-04, HNZK-06) were sequenced and analyzed. Phylogenetic analysis based on the complete genome, spike and nucleocapsid gene sequences revealed that the PDCoV Henan strains were closely related to other PDCoV reference strains that located in the Chinese clade. Furthermore, the phylogenetic analysis showed PDCoV CH-01 strain was closely related to CHN-HB-2014 strain and HKU15-44 strain, while the other PDCoV Henan strains were more related to PDCoV CHJXNI2 and CH-SXD1-2015 strains, indicating that the ancestor of these sequenced strains may different. These results would support the understanding of the prevalence and evolution characteristics of PDCoV in China.

negative: Scenario analysis of non-pharmaceutical interventions on global COVID-19 transmissions This paper introduces a dynamic panel SIR (DP-SIR) model to investigate the impact of non-pharmaceutical interventions (NPIs) on the COVID-19 transmission dynamics with panel data from 9 countries across the globe. By constructing scenarios with different combinations of NPIs, our empirical findings suggest that countries may avoid the lockdown policy with imposing school closure, mask wearing and centralized quarantine to reach similar outcomes on controlling the COVID-19 infection. Our results also suggest that, as of April 4th, 2020, certain countries such as the U.S. and Singapore may require additional measures of NPIs in order to control disease transmissions more effectively, while other countries may cautiously consider to gradually lift some NPIs to mitigate the costs to the overall economy.

negative: A complexity-based measure and its application to phylogenetic analysis In this article, we propose two well-defined distance metrics of biological sequences based on a universal complexity profile. To illustrate our metrics, phylogenetic trees of 18 Eutherian mammals from comparison of their mtDNA sequences and 24 coronaviruses using the whole genomes are constructed. The resulting monophyletic clusters agree well with the established taxonomic groups.

negative: Chapter 7 Long-Term Virus Evolution in Nature Abstract Viruses spread to give rise to epidemics and pandemics, and some key parameters that include virus and host population numbers determine virus persistence or extinction in nature. Viruses evolve at different rates of evolution depending on the polymerase copying fidelity during genome replication. Calculated rates of evolution in nature vary depending on the time interval between virus isolations. In particular, intra-host evolution is generally more rapid that inter-host evolution and several possible mechanisms for this difference are considered. The mechanisms by which the error-prone viruses evolve render very unlikely the operation of a molecular clock (constant rate of incorporation of mutations in the evolving genomes). Several computational methods are reviewed that permit the alignment of viral sequences and the establishment of phylogenetic relationships among viruses. The evolution of virus in the form of dynamic mutant clouds in each infected individual, together with multiple environmental influences, render the emergence and reemergence of viral pathogens an unpredictable event, another example of biological complexity.

negative: Genetic characterization of a novel picornavirus in Algerian bats: co-evolution analysis of bat-related picornaviruses Bats are reservoirs of numerous zoonotic viruses. The Picornaviridae family comprises important pathogens which may infect both humans and animals. In this study, a bat-related picornavirus was detected from Algerian Minioptreus schreibersii bats for the first time in the country. Molecular analyses revealed the new virus originates to the Mischivirus genus. In the operational use of the acquired sequence and all available data regarding bat picornaviruses, we performed a co-evolutionary analysis of mischiviruses and their hosts, to authentically reveal evolutionary patterns within this genus. Based on this analysis, we enlarged the dataset, and examined the co-evolutionary history of all bat-related picornaviruses including their hosts, to effectively compile all possible species jumping events during their evolution. Furthermore, we explored the phylogeny association with geographical location, host-genus and host-species in both data sets.

negative: Phylogenetic Insight into Zika and Emerging Viruses for a Perspective on Potential Hosts Global viral diversity is substantial, but viruses that contribute little to the public health burden or to agricultural damage receive minimal attention until a seemingly unimportant virus becomes a threat. The Zika virus (ZIKV) illustrated this, as there was limited information and awareness of the virus when it was identified as a public health emergency in February 2016. Predicting which virus may pose a future threat is difficult. This is in part because significant knowledge gaps in the basic biology and ecology of an emerging virus can impede policy development, delay decision making, and hinder public health action. We suggest using a phylogenetic framework of pathogens and their infected host species for insight into which animals may serve as reservoirs. For example, examining flaviviruses closely related to ZIKV, the phylogenetic framework indicates New World monkeys are the most likely candidates to be potential reservoirs for ZIKV. Secondarily, mammals that are in close proximity to humans should be considered because of the increased opportunity for pathogen exchange. The increase in human-mediated environmental change is accelerating the probability of another previously overlooked virus becoming a significant concern. By investing in basic science research and organizing our knowledge into an evolutionary framework, we will be better prepared to respond to the next emerging infectious disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10393-017-1237-x) contains supplementary material, which is available to authorized users.

negative: SARS-CoV2 (COVID-19) Structural/Evolution Dynamicome: Insights into functional evolution and human genomics The SARS-CoV-2 pandemic, starting in 2019, has challenged the speed at which labs perform science, ranging from discoveries of the viral composition to handling health outcomes in humans. The small ~30kb single-stranded RNA genome of Coronaviruses makes them adept at cross species spread and drift, increasing their probability to cause pandemics. However, this small genome also allows for a robust understanding of all proteins coded by the virus. We employed protein modeling, molecular dynamic simulations, evolutionary mapping, and 3D printing to gain a full proteome and dynamicome understanding of SARS-CoV-2. The Viral Integrated Structural Evolution Dynamic Database (VIStEDD) has been established (prokoplab.com/vistedd), opening future discoveries and educational usage. In this paper, we highlight VIStEDD usage for nsp6, Nucleocapsid (N), and Spike (S) surface glycoprotein. For both nsp6 and N we reveal highly conserved surface amino acids that likely drive protein-protein interactions. In characterizing viral S protein, we have developed a quantitative dynamics cross correlation matrix insight into interaction with the ACE2/SLC6A19 dimer complex. From this quantitative matrix, we elucidated 47 potential functional missense variants from population genomic databases within ACE2/SLC6A19/TMPRSS2, warranting genomic enrichment analyses in SARS-CoV-2 patients. Moreover, these variants have ultralow frequency, but can exist as hemizygous in males for ACE2, which falls on the X-chromosome. Two noncoding variants (rs4646118 and rs143185769) found in ~9% of African descent individuals for ACE2 may regulate expression and be related to increased susceptibility of African Americans to SARS-CoV-2. This powerful database of SARS-CoV-2 can aid in research progress in the ongoing pandemic.

negative: Whole-proteome phylogeny of large dsDNA viruses and parvoviruses through a composition vector method related to dynamical language model BACKGROUND: The vast sequence divergence among different virus groups has presented a great challenge to alignment-based analysis of virus phylogeny. Due to the problems caused by the uncertainty in alignment, existing tools for phylogenetic analysis based on multiple alignment could not be directly applied to the whole-genome comparison and phylogenomic studies of viruses. There has been a growing interest in alignment-free methods for phylogenetic analysis using complete genome data. Among the alignment-free methods, a dynamical language (DL) method proposed by our group has successfully been applied to the phylogenetic analysis of bacteria and chloroplast genomes. RESULTS: In this paper, the DL method is used to analyze the whole-proteome phylogeny of 124 large dsDNA viruses and 30 parvoviruses, two data sets with large difference in genome size. The trees from our analyses are in good agreement to the latest classification of large dsDNA viruses and parvoviruses by the International Committee on Taxonomy of Viruses (ICTV). CONCLUSIONS: The present method provides a new way for recovering the phylogeny of large dsDNA viruses and parvoviruses, and also some insights on the affiliation of a number of unclassified viruses. In comparison, some alignment-free methods such as the CV Tree method can be used for recovering the phylogeny of large dsDNA viruses, but they are not suitable for resolving the phylogeny of parvoviruses with a much smaller genome size.

negative: Phylogeny of Viruses☆ Abstract Biological species, including viruses, change through generations and over time in the process known as evolution. Viruses may evolve at high, uneven, and fluctuating rates among genome sites. The accumulated changes, through either mutation or recombination with other species, are first fixed in the genome of successful individuals that give rise to genetic lineages. The relationship between biological lineages related by common descent is called ‘phylogeny’. For inferring phylogeny, the differences between aligned sequences of genomes and proteins are quantified and depicted in the form of a tree, in which contemporary species and their intermediate and common ancestors occupy, respectively, the terminal nodes, internal nodes, and the root. The tree is characterized by a topology, length of branches, shape, and the root position. A complex mathematical apparatus has been developed for phylogeny inference that can evaluate inter-species differences, facilitate tree building and comparison of trees, and assess the fit between data and tree through, typically, computationally intensive calculations. A reconstructed tree is an approximation of the true phylogeny that practically remains unknown. The phylogenetic analysis is used in applied and fundamental virus research, including epidemiology, diagnostics, forensic studies, phylogeography, evolutionary studies, and virus taxonomy. It can provide an evolutionary perspective on variation of any trait that can be measured for a group of viruses.

negative: Complete Genome Sequence of Avian Coronavirus Strain GA08 (GI-27 Lineage) Avian coronavirus, also known as infectious bronchitis virus, is a highly contagious respiratory pathogen of chickens that is responsible for major economic losses to the poultry industry around the globe. Here, we report the complete genome sequence of strain GA08 of the GI-27 lineage, isolated from a fecal sample from a broiler chicken collected in Georgia in 2015.

negative: Coronaviruses and SARS-COV-2 Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 (COVID-19). COVID-19’s first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.

negative: Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

negative: EDGE COVID-19: A Web Platform to generate submission-ready genomes for SARS-CoV-2 sequencing efforts Genomics has become a pivotal technology for understanding emerging outbreaks of infectious diseases. A wide range of technologies and strategies for pathogen genome enrichment and sequencing are being used by laboratories and institutes, resulting in different analytical procedures for generating genome sequences and resulting in non-standard entries of varying quality into public repositories. A standardized analytical process for genome sequence generation, particularly for outbreaks such as the ongoing COVID-19 pandemic, are critical to provide scientists with a solid genomic basis for epidemiology analyses and for making well-informed decisions. To address this challenge, we have developed a web platform (EDGE COVID-19) to run standardized workflows for reference-based genome assembly and to perform further analysis of Illumina or Oxford Nanopore Technologies data for SARS-CoV-2 genome sequencing projects. Given a raw Illumina or Oxford Nanopore FASTQ read file, this web platform automates the production of a SARS-CoV-2 genome that is ready for submission to GISAID or GenBank. EDGE COVID-19 is open-source software that is available as a web application for public use at https://covid19.edgebioinformatics.org, and as a Docker container at https://hub.docker.com/r/bioedge/edge-covid19 for local installation.

negative: Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Northern Germany Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany.

negative: Whole-genome characterization of Uruguayan strains of avian infectious bronchitis virus reveals extensive recombination between the two major South American lineages Abstract Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus that causes one of the most persistent respiratory diseases in poultry. The virus is classified in genotypes and lineages with different epidemiological relevance. Two lineages of the GI genotype (11 and 16) have been widely circulating for decades in South America. GI-11 is an exclusive South American lineage while the GI-16 lineage is distributed in Asia, Europe and South America. Here, we obtained the whole genome of two Uruguayan strains of the GI-11 and GI-16 lineages using Illumina high-throughput sequencing. The strains here sequenced are the first obtained in South America for the infectious bronchitis virus and provide new insights into the origin, spreading and evolution of viral variants. The complete genome of the GI-11 and GI-16 strains have 27,621 and 27,638 nucleotides, respectively, and possess the same genomic organization. Phylogenetic incongruence analysis reveals that both strains have a mosaic genome that arose by recombination between Euro Asiatic strains of the GI-16 lineage and ancestral South American GI-11 viruses. The recombination occurred in South America and produced two viral variants that have retained the full-length S1 sequences of the parental lineages but are extremely similar in the rest of their genomes. These recombinant virus have been extraordinary successful, persisting in the continent for several years with a notorious wide geographic distribution. Our findings reveal a singular viral dynamics and emphasize the importance of complete genomic characterization to understand the emergence and evolutionary history of viral variants.

negative: Racial and Ethnic Disparities in SARS-CoV-2 Pandemic: Analysis of a COVID-19 Observational Registry for a Diverse U.S. Metropolitan Population Importance: Despite emerging reports of poor COVID-19 outcomes among African Americans, data on race and ethnic susceptibility to SARS-CoV-2 infection are limited. Objective: To determine socio-demographic factors associated with higher likelihood of SARS-CoV-2 infection. To explore mediating pathways for race disparities in the SARS-CoV-2 pandemic. Design: Cross sectional analysis of COVID-19 Surveillance and Outcomes Registry (CURATOR). Multivariable logistic regression models were fitted to provide likelihood estimates (adjusted Odds Ratios: aOR, 95% confidence intervals: CI) of positive SARS-CoV-2 test. Structural Equation Modeling (SEM) framework was utilized to explore three mediation pathways (low income, high population density, high comorbidity burden) for association between African American race and SARS-CoV-2 infection. Setting: A large healthcare system comprising of one central tertiary care, seven large community hospitals and an expansive ambulatory and emergency care network in the Greater Houston area. Participants: Individuals of all ages, races, ethnicities and sex tested for SARS-CoV-2. Exposure: Socio-demographic (age, sex, race, ethnicity, household income, residence population density) and comorbidity (hypertension, diabetes, obesity, cardiac disease) factors. Main Outcome: Positive reverse transcriptase polymerized chain reaction test for SARS-CoV-2. Results: Among 4,513 tested individuals, 754 (16.7%) tested positive. Overall mean (SD) age was 50.6 (18.9) years, 62% females and 26% were African American. African American race was associated with higher comorbidity burden, lower socio-economic status, and higher population density residence. In the fully adjusted model, African American race (vs. White; aOR, CI: 1.84, 1.49-2.27) and Hispanic ethnicity (vs. non-Hispanic; aOR, CI: 1.70, 1.35-2.14) had a higher likelihood of infection. Older individuals and males were also at a higher risk of SARS-CoV-2 infection. The SEM framework demonstrated a statistically significant (p = 0.008) indirect effect of African American race on SARS-CoV-2 infection mediated via a pathway that included residence in densely populated zip code. Conclusions and Relevance: There is strong evidence of race and ethnic disparities in the SARS-CoV-2 pandemic potentially mediated through unique social determinants of health.

negative: Potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill COVID-19

negative: Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans.

negative: Molecular identification of Betacoronavirus in bats from Sardinia (Italy): first detection and phylogeny Bats may be natural reservoirs for a large variety of emerging viruses, including mammalian coronaviruses (CoV). The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) in humans, with evidence that these viruses may have their ancestry in bats, highlights the importance of virus surveillance in bat populations. Here, we report the identification and molecular characterization of a bat β-Coronavirus, detected during a viral survey carried out on different bat species in the island of Sardinia (Italy). Cutaneous, oral swabs, and faecal samples were collected from 46 bats, belonging to 15 different species, and tested for viral presence. Coronavirus RNA was detected in faecal samples from three different species: the greater horseshoe bat (Rhinolophus ferrumequinum), the brown long-eared bat (Plecotus auritus), and the European free-tailed bat (Tadarida teniotis). Phylogenetic analyses based on RNA-dependent RNA polymerase (RdRp) sequences assigned the detected CoV to clade 2b within betacoronaviruses, clustering with SARS-like bat CoVs previously reported. These findings point to the need for continued surveillance of bat CoV circulating in Sardinian bats, and extend the current knowledge on CoV ecology with novel sequences detected in bat species not previously described as β-Coronavirus hosts.

negative: Diversity and Epidemiology of Mokola Virus Mokola virus (MOKV) appears to be exclusive to Africa. Although the first isolates were from Nigeria and other Congo basin countries, all reports over the past 20 years have been from southern Africa. Previous phylogenetic studies analyzed few isolates or used partial gene sequence for analysis since limited sequence information is available for MOKV and the isolates were distributed among various laboratories. The complete nucleoprotein, phosphoprotein, matrix and glycoprotein genes of 18 MOKV isolates in various laboratories were sequenced either using partial or full genome sequencing using pyrosequencing and a phylogenetic analysis was undertaken. The results indicated that MOKV isolates from the Republic of South Africa, Zimbabwe, Central African Republic and Nigeria clustered according to geographic origin irrespective of the genes used for phylogenetic analysis, similar to that observed with Lagos bat virus. A Bayesian Markov-Chain-Monte-Carlo- (MCMC) analysis revealed the age of the most recent common ancestor (MRCA) of MOKV to be between 279 and 2034 years depending on the genes used. Generally, all MOKV isolates showed a similar pattern at the amino acid sites considered influential for viral properties.

negative: SARS-CoV-2 contributes to altering the post-transcriptional regulatory networks across human tissues by sponging RNA binding proteins and micro-RNAs The outbreak of a novel coronavirus SARS-CoV2 responsible for COVID-19 pandemic has caused worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2 mediated alteration on post-transcriptional gene regulation across human tissues remains elusive. In this study, we systematically dissected the crosstalk and dysregulation of human post-transcriptional regulatory networks governed by RNA binding proteins (RBPs) and micro-RNAs (miRs), due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2 encoded proteins directly interact with 51 human RBPs of which majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed functional analysis of differentially expressed genes in mock treated versus SARS-CoV-2 infected lung cells that revealed an enrichment for immune response, cytokine mediated signaling, and metabolism associated genes. This study also characterized the alternative splicing events in SARS-CoV-2 infected cells compared to control demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to viral infection. Motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed an enrichment for RBPs such as SRSFs, PCBPs, ELAVs and HNRNPs illustrating the sponging of RBPs by SARS-CoV-2 genome. Similar analysis to study the interactions of miRs with SARS-CoV-2 revealed the potential for several miRs to be sponged, suggesting that these interactions may contribute to altered pos-transcriptional regulation across human tissues. Given the need to understand the interactions of SARS-CoV-2 with key pos-transcriptional regulators in the human genome, this study provides a systematic analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs, across tissues types during SARS-CoV2 infection.

negative: A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19 PURPOSE: COVID-19 (coronavirus disease 2019) is a public health emergency of international concern. As of this time, there is no known effective pharmaceutical treatment, although it is much needed for patient contracting the severe form of the disease. The aim of this systematic review was to summarize the evidence regarding chloroquine for the treatment of COVID-19. METHODS: PubMed, EMBASE, and three trial Registries were searched for studies on the use of chloroquine in patients with COVID-19. RESULTS: We included six articles (one narrative letter, one in-vitro study, one editorial, expert consensus paper, two national guideline documents) and 23 ongoing clinical trials in China. Chloroquine seems to be effective in limiting the replication of SARS-CoV-2 (virus causing COVID-19) in vitro. CONCLUSIONS: There is rationale, pre-clinical evidence of effectiveness and evidence of safety from long-time clinical use for other indications to justify clinical research on chloroquine in patients with COVID-19. However, clinical use should either adhere to the Monitored Emergency Use of Unregistered Interventions (MEURI) framework or be ethically approved as a trial as stated by the World Health Organization. Safety data and data from high-quality clinical trials are urgently needed.

negative: Five decades of research on foreign direct investment by MNEs: An overview and research agenda Abstract Despite the significance attached to foreign direct investment (FDI) by Multinational enterprises (MNEs), there are is no comprehensive review of the FDI literature. Moreover, those that have been published, focus on subsets of FDI. This review systematically examines the empirical as well as theoretical research on FDI through an analysis of 500 articles published during the last five decades. Theoretical models, methods, context, and contributions to scholarship were reviewed. We strive to highlight the key theories, paradigms, and articles and provide directions for future research. We conclude that FDI has evolved as the most significant area of international business.

negative: What about the original antigenic sin of the humans versus SARS-CoV-2?

negative: Ethical considerations in global HIV phylogenetic research. Phylogenetic analysis of pathogens is an increasingly powerful way to reduce the spread of epidemics, including HIV. As a result, phylogenetic approaches are becoming embedded in public health and research programmes, as well as outbreak responses, presenting unique ethical, legal, and social issues that are not adequately addressed by existing bioethics literature. We formed a multidisciplinary working group to explore the ethical issues arising from the design of, conduct in, and use of results from HIV phylogenetic studies, and to propose recommendations to minimise the associated risks to both individuals and groups. We identified eight key ethical domains, within which we highlighted factors that make HIV phylogenetic research unique. In this Review, we endeavoured to provide a framework to assist researchers, public health practitioners, and funding institutions to ensure that HIV phylogenetic studies are designed, done, and disseminated in an ethical manner. Our conclusions also have broader relevance for pathogen phylogenetics.

negative: In memory of Patrick Manson, founding father of tropical medicine and the discovery of vector-borne infections Patrick Manson, a clinician-scientist serving in China (1866–1889), discovered that many tropical infectious diseases require a vector peculiar to warm climate for person to person transmission. He demonstrated the nocturnal periodicity of microfilariae in the blood of patients with elephantiasis. These microfilariae undergo metamorphosis when ingested by the mosquito acting as the vector for the completion of their life cycle. Furthermore, he demonstrated the linkage between the lung fluke and endemic haemoptysis by finding operculated eggs in patients' sputa. He predicted that the miracidium from hatched eggs uses crustaceans, such as fresh-water snails found at tropical conditions, as the intermediate hosts in the life cycle of many trematodes. His vector hypothesis leads to vector control which is now the cornerstone for the World Health Organization's programme for the elimination/control of lymphatic filariasis, dracunculiasis and malaria. Before leaving China, he established the Alice Memorial Hospital, the Hong Kong College of Medicine for Chinese (the forerunner of the University of Hong Kong), and the Hong Kong Medical Society for medical service and education. He also incepted the Hong Kong Dairy Farm for supplying hygienic milk affordable by pregnant women, children and patients.

negative: Coronavirus surveillance of wildlife in the Lao People’s Democratic Republic detects viral RNA in rodents Coronaviruses can become zoonotic, as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia increases the risk for such incidents. We sampled and tested rodents (851) and other mammals and found betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters and are closely related to those of known rodent coronaviruses detected in the region and distantly related to those of human coronaviruses OC43 and HKU1. Considering the close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00705-020-04683-7) contains supplementary material, which is available to authorized users.

negative: Assessing the fear of COVID-19 among different populations: A response to Ransing et al. (2020)

negative: A48 Identification and full-genome characterization of Alpha- and Beta-Coronaviruses viruses from bats in Italy Bats are the natural reservoir of Coronaviruses (CoVs). Human CoVs cause mild respiratory diseases worldwide, but, in the last decade, two Beta-CoVs [Middle East respiratory syndrome (MERS)-CoV and severe acute respiratory syndrome] caused thousands of deaths and cases worldwide. Phylogenetic analysis suggested the evolutionary origin of mammalian CoVs is derived from bats. In this study, we characterized three Alpha-CoVs and two Beta-CoVs demonstrating the circulation of bat strains in Italy. Isolates were sequenced using a next-generation sequencing approach and genomes reconstructed using the online tool Galaxy Aries. Phylogenetic analyses were conducted using MEGA7 and MrBayes. Similarity plots were generated using SSE v1.2. The structure of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank. Bioinformatics analysis permitted the identification of 2 Beta-CoV complete genomes of 30 kb and three Alpha-CoV of 28 kb (named BatCoV-ITA1-5). BatCoV-ITA1 and 2 formed a monophyletic group with MERS-CoV sequences. The comparison of the concatenated domains within ORF1ab confirmed their classification into the MERS-CoV species. The 3D structure of RBD of Italian strains showed two amino acid deletions located in a region corresponding to the external subdomain of MERS-RBD. BatCoV-Ita3 and BatCoV-Ita4/5 were classified into two novel Alpha-CoV species by comparison of concatenated domains within ORF1ab. Due to the high divergence with the Alpha human spike protein strains, it was impossible to establish the protein structure and the potential affinity to human receptor. The Italian strains showed the typical organization of Alpha and Beta-CoVs. We reported two Beta-CoVs closely related to MERS-CoVs from bats belonging to common Italian species (Pipistrellus kuhlii and Hypsugo savii). The analysis of the RBD in the spike protein indicates significant differences from human RBD known to date. The three Alpha-CoV strains were classified into two novel species, confirming the high heterogeneity of CoV strains in bats. Although the studies conducted cannot confirm a risk for humans, surveillance studies are needed to investigate the genetic diversity of CoVs in bats. Because this exceeds what is known for other hosts, it is compatible with bats being the major reservoir of mammalian CoVs.

negative: The Structural Characterization and Antigenicity of the S Protein of SARS-CoV The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phylogenetic analysis reveals several conserved regions that might be potent drug targets.

negative: A Rare Deletion in SARS-CoV-2 ORF6 Dramatically Alters the Predicted Three-Dimensional Structure of the Resultant Protein The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion (Δ27,264-27,290) within ORF6, predicted to result in a 9 aa deletion (ΔFKVSIWNLD) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6Δ22-30), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6Δ22-30 N-terminus, possibly leading to the ablation of its native function.

negative: Structural Organization of the Genome of SARS-Associated Coronavirus (Strain SoD) Isolated on the Territory of the Russian Federation Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.

negative: SARS-CoV-2 is an appropriate name for the new coronavirus

negative: Characterization of the substitution hotspots in SARS-CoV-2 genome using BioAider and detection of a SR-rich region in N protein providing further evidence of its animal origin The novel human coronavirus (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic worldwide. The increasing sequencing data have shown abundant single nucleotide variations in SARS-CoV-2 genome. However, it is difficult to quickly analyze genomic variation and screen key mutations of SARS-CoV-2. In this study, we developed a visual program, named BioAider, for quick and convenient sequence annotation and mutation analysis on multiple genome-sequencing data. Using BioAider, we conducted a comprehensive genome variation analysis on 3,240 sequences of SARS-CoV-2 genome. Herein, we detected 14 substitution hotspots within SARS-CoV-2 genome, including 10 non-synonymous and 4 synonymous ones. Among these hotspots, NSP13-Y541C was predicted to be a crucial substitution which might affect the unwinding activity of NSP13, a key protein for viral replication. Besides, we also found 3 groups of potentially linked substitution hotspots which were worth further study. In particular, we discovered a SR-rich region (aa 184-204) on the N protein of SARS-CoV-2 distinct from SARS-CoV, indicating more complex replication mechanism and unique N-M interaction of SARS-CoV-2. Interestingly, the quantity of SRXX repeat fragments in the SR-rich region well reflected the evolutionary relationship among SARS-CoV-2 and SARS-CoV-2 related animal coronaviruses, providing further evidence of its animal origin. Overall, we developed an efficient tool for rapid identification of mutations, identified substitution hotspots in SARS-CoV-2 genomes, and detected a distinctive polymorphism SR-rich region in N protein. This tool and the detected hotspots could facilitate the viral genomic study and may contribute for screening antiviral target sites.

negative: Molecular mechanism of evolution and human infection with the novel coronavirus (2019-nCoV) Since December, 2019, an outbreak of pneumonia caused by the new coronavirus (2019-nCoV) has hit the city of Wuhan in the Hubei Province. With the continuous development of the epidemic, it has become a national public health crisis and calls for urgent antiviral treatments or vaccines. The spike protein on the coronavirus envelope is critical for host cell infection and virus vitality. Previous studies showed that 2019-nCoV is highly homologous to human SARS-CoV and attaches host cells though the binding of the spike receptor binding domain (RBD) domain to the angiotensin-converting enzyme II (ACE2). However, the molecular mechanisms of 2019-nCoV binding to human ACE2 and evolution of 2019-nCoV remain unclear. In this study, we have extensively studied the RBD-ACE2 complex, spike protein, and free RBD systems of 2019-nCoV and SARS-CoV using protein-protein docking and molecular dynamics (MD) simulations. It was shown that the RBD-ACE2 binding free energy for 2019-nCoV is significantly lower than that for SARS-CoV, which is consistent with the fact that 2019-nCoV is much more infectious than SARS-CoV. In addition, the spike protein of 2019-nCoV shows a significantly lower free energy than that of SARS-CoV, suggesting that 2019-nCoV is more stable and able to survive a higher temperature than SARS-CoV. This may also provide insights into the evolution of 2019-nCoV because SARS-like coronaviruses are thought to have originated in bats that are known to have a higher body-temperature than humans. It was also revealed that the RBD of 2019-nCoV is much more flexible especially near the binding site and thus will have a higher entropy penalty upon binding ACE2, compared to the RBD of SARS-CoV. That means that 2019-nCoV will be much more temperature-sensitive in terms of human infection than SARS-CoV. With the rising temperature, 2019-nCoV is expected to decrease its infection ability much faster than SARS-CoV, and get controlled more easily. The present findings are expected to be helpful for the disease prevention and control as well as drug and vaccine development of 2019-nCoV.

negative: A COMPREHENSIVE ANALYSIS OF GENOME COMPOSITION AND CODON USAGE PATTERNS OF EMERGING CORONAVIRUSES Abstract An outbreak of atypical pneumonia caused by a novel Betacoronavirus (βCoV), named SARS-CoV-2 has been declared a public health emergency of international concern by the World Health Organization. In order to gain insight into the emergence, evolution and adaptation of SARS-CoV-2 viruses, a comprehensive analysis of genome composition and codon usage of βCoV circulating in China was performed. A biased nucleotide composition was found for SARS-CoV-2 genome. This bias in genomic composition is reflected in its codon and amino acid usage patterns. The overall codon usage in SARS-CoV-2 is similar among themselves and slightly biased. Most of the highly frequent codons are A- and U-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. Significant differences in relative synonymous codon usage frequencies among SARS-CoV-2 and human cells were found. These differences are due to codon usage preferences.

negative: Coronavirus diversity, phylogeny and interspecies jumping. The SARS epidemic has boosted interest in research on coronavirus biodiversity and genomics. Before 2003, there were only 10 coronaviruses with complete genomes available. After the SARS epidemic, up to December 2008, there was an addition of 16 coronaviruses with complete genomes sequenced. These include two human coronaviruses (human coronavirus NL63 and human coronavirus HKU1), 10 other mammalian coronaviruses [bat SARS coronavirus, bat coronavirus (bat-CoV) HKU2, bat-CoV HKU4, bat-CoV HKU5, bat-CoV HKU8, bat-CoV HKU9, bat-CoV 512/2005, bat-CoV 1A, equine coronavirus, and beluga whale coronavirus] and four avian coronaviruses (turkey coronavirus, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13). Two novel subgroups in group 2 coronavirus (groups 2c and 2d) and two novel subgroups in group 3 coronavirus (groups 3b and 3c) have been proposed. The diversity of coronaviruses is a result of the infidelity of RNA-dependent RNA polymerase, high frequency of homologous RNA recombination, and the large genomes of coronaviruses. Among all hosts, the diversity of coronaviruses is most evidenced in bats and birds, which may be a result of their species diversity, ability to fly, environmental pressures, and habits of roosting and flocking. The present evidence supports that bat coronaviruses are the gene pools of group 1 and 2 coronaviruses, whereas bird coronaviruses are the gene pools of group 3 coronaviruses. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed, which were results of recent interspecies jumping and may be the cause of disastrous outbreaks of zoonotic diseases.

negative: Genotyping SARS-CoV-2 through an interactive web application

negative: SYSGENET: a meeting report from a new European network for systems genetics The first scientific meeting of the newly established European SYSGENET network took place at the Helmholtz Centre for Infection Research (HZI) in Braunschweig, April 7-9, 2010. About 50 researchers working in the field of systems genetics using mouse genetic reference populations (GRP) participated in the meeting and exchanged their results, phenotyping approaches, and data analysis tools for studying systems genetics. In addition, the future of GRP resources and phenotyping in Europe was discussed.

negative: The Phylogeography of MERS-CoV in Hospital Outbreak-Associated Cases Compared to Sporadic Cases in Saudi Arabia This study compared the phylogeography of MERS-CoV between hospital outbreak-associated cases and sporadic cases in Saudi Arabia. We collected complete genome sequences from human samples in Saudi Arabia and data on the multiple risk factors of human MERS-CoV in Saudi Arabia reported from 2012 to 2018. By matching each sequence to human cases, we identified isolates as hospital outbreak-associated cases or sporadic cases. We used Bayesian phylogenetic methods including temporal, discrete trait analysis and phylogeography to uncover transmission routes of MERS-CoV isolates between hospital outbreaks and sporadic cases. Of the 120 sequences collected between 19 June 2012 and 23 January 2017, there were 64 isolates from hospital outbreak-associated cases and 56 from sporadic cases. Overall, MERS-CoV is fast evolving at 7.43 × 10(−4) substitutions per site per year. Isolates from hospital outbreaks showed unusually fast evolutionary speed in a shorter time-frame than sporadic cases. Multiple introductions of different MERS-CoV strains occurred in three separate hospital outbreaks. MERS-CoV appears to be mutating in humans. The impact of mutations on viruses transmissibility in humans is unknown.

negative: The emergence of SARS-CoV-2 in Europe and the US Accurate understanding of the global spread of emerging viruses is critically important for public health response and for anticipating and preventing future outbreaks. Here, we elucidate when, where and how the earliest sustained SARS-CoV-2 transmission networks became established in Europe and the United States (US). Our results refute prior findings erroneously linking cases in January 2020 with outbreaks that occurred weeks later. Instead, rapid interventions successfully prevented onward transmission of those early cases in Germany and Washington State. Other, later introductions of the virus from China to both Italy and Washington State founded the earliest sustained European and US transmission networks. Our analyses reveal an extended period of missed opportunity when intensive testing and contact tracing could have prevented SARS-CoV-2 from becoming established in the US and Europe.

negative: Social Media as a Sensor of Air Quality and Public Response in China BACKGROUND: Recent studies have demonstrated the utility of social media data sources for a wide range of public health goals, including disease surveillance, mental health trends, and health perceptions and sentiment. Most such research has focused on English-language social media for the task of disease surveillance. OBJECTIVE: We investigated the value of Chinese social media for monitoring air quality trends and related public perceptions and response. The goal was to determine if this data is suitable for learning actionable information about pollution levels and public response. METHODS: We mined a collection of 93 million messages from Sina Weibo, China’s largest microblogging service. We experimented with different filters to identify messages relevant to air quality, based on keyword matching and topic modeling. We evaluated the reliability of the data filters by comparing message volume per city to air particle pollution rates obtained from the Chinese government for 74 cities. Additionally, we performed a qualitative study of the content of pollution-related messages by coding a sample of 170 messages for relevance to air quality, and whether the message included details such as a reactive behavior or a health concern. RESULTS: The volume of pollution-related messages is highly correlated with particle pollution levels, with Pearson correlation values up to .718 (n=74, P<.001). Our qualitative results found that 67.1% (114/170) of messages were relevant to air quality and of those, 78.9% (90/114) were a firsthand report. Of firsthand reports, 28% (32/90) indicated a reactive behavior and 19% (17/90) expressed a health concern. Additionally, 3 messages of 170 requested that action be taken to improve quality. CONCLUSIONS: We have found quantitatively that message volume in Sina Weibo is indicative of true particle pollution levels, and we have found qualitatively that messages contain rich details including perceptions, behaviors, and self-reported health effects. Social media data can augment existing air pollution surveillance data, especially perception and health-related data that traditionally requires expensive surveys or interviews.

negative: Pulmonary cowpox in cats: five cases. CASE SERIES SUMMARY This case series documents five cases of pneumonia (with pleural effusion in three cases) caused by cowpox virus (CPxV) in domestic cats. Predisposition to pneumonia may have resulted from mixed infections in two cases (feline herpesvirus and Bordetella bronchiseptica in one cat, and Mycoplasma species in the other). RELEVANCE AND NOVEL INFORMATION As well as diagnostic confirmation by previously described methods of virus isolation from skin lesions, and demonstration of pox virions in skin samples using electron microscopy and inclusion bodies in histological preparations, this is the first report of diagnosis by virus isolation from bronchoalveolar lavage fluid or pleural fluid, and demonstration of inclusion bodies in cytological preparations. This is also the first series to report treatment with interferon omega (IFN-ω). Two cats survived, both of which had been treated with IFN-ω. As CPxV represents a serious zoonotic risk it is an important differential diagnosis of pneumonia in cats.

negative: Drug sales data analysis for outbreak detection of infectious diseases: a systematic literature review BACKGROUND: This systematic literature review aimed to summarize evidence for the added value of drug sales data analysis for the surveillance of infectious diseases. METHODS: A search for relevant publications was conducted in Pubmed, Embase, Scopus, Cochrane Library, African Index Medicus and Lilacs databases. Retrieved studies were evaluated in terms of objectives, diseases studied, data sources, methodologies and performance for real-time surveillance. Most studies compared drug sales data to reference surveillance data using correlation measurements or indicators of outbreak detection performance (sensitivity, specificity, timeliness of the detection). RESULTS: We screened 3266 articles and included 27 in the review. Most studies focused on acute respiratory and gastroenteritis infections. Nineteen studies retrospectively compared drug sales data to reference clinical data, and significant correlations were observed in 17 of them. Four studies found that over-the-counter drug sales preceded clinical data in terms of incidence increase. Five studies developed and evaluated statistical algorithms for selecting drug groups to monitor specific diseases. Another three studies developed models to predict incidence increase from drug sales. CONCLUSIONS: Drug sales data analyses appear to be a useful tool for surveillance of gastrointestinal and respiratory disease, and OTC drugs have the potential for early outbreak detection. Their utility remains to be investigated for other diseases, in particular those poorly surveyed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12879-014-0604-2) contains supplementary material, which is available to authorized users.

negative: Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses Seven coronaviruses (CoVs) have been isolated from humans so far. Among them, three emerging pathogenic CoVs, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and a newly identified CoV (2019-nCoV), once caused or continue to cause severe infections in humans, posing significant threats to global public health. SARS-CoV infection in humans (with about 10% case fatality rate) was first reported from China in 2002, while MERS-CoV infection in humans (with about 34.4% case fatality rate) was first reported from Saudi Arabia in June 2012. 2019-nCoV was first reported from China in December 2019, and is currently infecting more than 70000 people (with about 2.7% case fatality rate). Both SARS-CoV and MERS-CoV are zoonotic viruses, using bats as their natural reservoirs, and then transmitting through intermediate hosts, leading to human infections. Nevertheless, the intermediate host for 2019-nCoV is still under investigation and the vaccines against this new CoV have not been available. Although a variety of vaccines have been developed against infections of SARS-CoV and MERS-CoV, none of them has been approved for use in humans. In this review, we have described the structure and function of key proteins of emerging human CoVs, overviewed the current vaccine types to be developed against SARS-CoV and MERS-CoV, and summarized recent advances in subunit vaccines against these two pathogenic human CoVs. These subunit vaccines are introduced on the basis of full-length spike (S) protein, receptor-binding domain (RBD), non-RBD S protein fragments, and non-S structural proteins, and the potential factors affecting these subunit vaccines are also illustrated. Overall, this review will be helpful for rapid design and development of vaccines against the new 2019-nCoV and any future CoVs with pandemic potential. This review was written for the topic of Antivirals for Emerging Viruses: Vaccines and Therapeutics in the Virology section of Frontiers in Microbiology.

negative: Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (MED1, MED9, MED12L, MED19), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STAT1 were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.

negative: Transmission patterns of smallpox: systematic review of natural outbreaks in Europe and North America since World War II BACKGROUND: Because smallpox (variola major) may be used as a biological weapon, we reviewed outbreaks in post-World War II Europe and North America in order to understand smallpox transmission patterns. METHODS: A systematic review was used to identify papers from the National Library of Medicine, Embase, Biosis, Cochrane Library, Defense Technical Information Center, WorldCat, and reference lists of included publications. Two authors reviewed selected papers for smallpox outbreaks. RESULTS: 51 relevant outbreaks were identified from 1,389 publications. The median for the effective first generation reproduction rate (initial R) was 2 (range 0–38). The majority outbreaks were small (less than 5 cases) and contained within one generation. Outbreaks with few hospitalized patients had low initial R values (median of 1) and were prolonged if not initially recognized (median of 3 generations); outbreaks with mostly hospitalized patients had higher initial R values (median 12) and were shorter (median of 3 generations). Index cases with an atypical presentation of smallpox were less likely to have been diagnosed with smallpox; outbreaks in which the index case was not correctly diagnosed were larger (median of 27.5 cases) and longer (median of 3 generations) compared to outbreaks in which the index case was correctly diagnosed (median of 3 cases and 1 generation). CONCLUSION: Patterns of spread during Smallpox outbreaks varied with circumstances, but early detection and implementation of control measures is a most important influence on the magnitude of outbreaks. The majority of outbreaks studied in Europe and North America were controlled within a few generations if detected early.

negative: Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) samples from field cases in Fujian, China The outbreak of porcine epidemic diarrhea virus (PEDV) has been a big problem of swine industry in China in recent years. In this study, we investigated molecular diversity, phylogenetic relationships, and protein characterization of Fujian field samples with other PEDV reference strains. Sequence analysis of the S1 and sM genes showed that each sample had unique characteristics, and the sample P55 may be differentiated from the others by the unique deletions and insertions of sM gene. Phylogenetic analysis based on S1 or sM gene, which have high levels of variations, indicated that each sample was related to the specific reference strain, and this finding was consistent with the protein characterization prediction analysis. The study is useful to better understand the prevalence of PEDV and its prevention and control in Fujian.

negative: COVID-19 Virulence in Aged Patients Might Be Impacted by the Host Cellular MicroRNAs Abundance/Profile The World health organization (WHO) declared Coronavirus disease 2019 (COVID-19) a global pandemic and a severe public health crisis. Drastic measures to combat COVID-19 are warranted due to its contagiousness and higher mortality rates, specifically in the aged patient population. At the current stage, due to the lack of effective treatment strategies for COVID-19 innovative approaches need to be considered. It is well known that host cellular miRNAs can directly target both viral 3'UTR and coding region of the viral genome to induce the antiviral effect. In this study, we did in silico analysis of human miRNAs targeting SARS (4 isolates) and COVID-19 (29 recent isolates from different regions) genome and correlated our findings with aging and underlying conditions. We found 848 common miRNAs targeting the SARS genome and 873 common microRNAs targeting the COVID-19 genome. Out of a total of 848 miRNAs from SARS, only 558 commonly present in all COVID-19 isolates. Interestingly, 315 miRNAs are unique for COVID-19 isolates and 290 miRNAs unique to SARS. We also noted that out of 29 COVID-19 isolates, 19 isolates have identical miRNA targets. The COVID-19 isolates, Netherland (EPI_ISL_422601), Australia (EPI_ISL_413214), and Wuhan (EPI_ISL_403931) showed six, four, and four unique miRNAs targets, respectively. Furthermore, GO, and KEGG pathway analysis showed that COVID-19 targeting human miRNAs involved in various age-related signaling and diseases. Recent studies also suggested that some of the human miRNAs targeting COVID-19 decreased with aging and underlying conditions. GO and KEGG identified impaired signaling pathway may be due to low abundance miRNA which might be one of the contributing factors for the increasing severity and mortality in aged individuals and with other underlying conditions. Further, in vitro and in vivo studies are needed to validate some of these targets and identify potential therapeutic targets.

negative: Critical Differences between the Binding Features of the Spike Proteins of SARS-CoV-2 and SARS-CoV [Image: see text] The COVID-19 caused by SARS-CoV-2 has spread globally and caused tremendous loss of lives and properties, and it is of utmost urgency to understand its propagation process and to find ways to slow down the epidemic. In this work, we used a coarse-grained model to calculate the binding free energy of SARS-CoV-2 or SARS-CoV to their human receptor ACE2. The investigation of the free energy contribution of the interacting residues indicates that the residues located outside the receptor binding domain are the source of the stronger binding of the novel virus. Thus, the current results suggest that the essential evolution of SARS-CoV-2 happens remotely from the binding domain at the spike protein trimeric body. Such evolution may facilitate the conformational change and the infection process that occurs after the virus is bound to ACE2. By studying the binding pattern between SARS-CoV antibody m396 and SARS-CoV-2, it is found that the remote energetic contribution is missing, which might explain the absence of cross-reactivity of such antibodies.

negative: Novel SARS-like Betacoronaviruses in Bats, China, 2011 To clarify the evolutionary relationships among betavoronaviruses that infect bats, we analyzed samples collected during 2010–2011 from 14 insectivorous bat species in China. We identified complete genomes of 2 novel betacoronaviruses in Rhinolophus pusillus and Chaerephon plicata bats, which showed close genetic relationships with severe acute respiratory syndrome coronaviruses.

negative: Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003 Summary Background An epidemic of severe acute respiratory syndrome (SARS) has been associated with an outbreak of atypical pneumonia originating in Guangdong Province, People's Republic of China. We aimed to identify the causative agent in the Guangdong outbreak and describe the emergence and spread of the disease within the province. Methods We analysed epidemiological information and collected serum and nasopharyngeal aspirates from patients with SARS in Guangdong in mid-February, 2003. We did virus isolation, serological tests, and molecular assays to identify the causative agent. Findings SARS had been circulating in other cities of Guangdong Province for about 2 months before causing a major outbreak in Guangzhou, the province's capital. A novel coronavirus, SARS coronavirus (CoV), was isolated from specimens from three patients with SARS. Viral antigens were also directly detected in nasopharyngeal aspirates from these patients. 48 of 55 (87%) patients had antibodies to SARS CoV in their convalescent sera. Genetic analysis showed that the SARS CoV isolates from Guangzhou shared the same origin with those in other countries, and had a phylogenetic pathway that matched the spread of SARS to the other parts of the world. Interpretation SARS CoV is the infectious agent responsible for the epidemic outbreak of SARS in Guangdong. The virus isolated from patients in Guangdong is the prototype of the SARS CoV in other regions and countries.

negative: Molecular Epidemiology of Severe Acute Respiratory Syndrome–Associated Coronavirus Infections in Taiwan Background In 2003, Taiwan experienced a series of outbreaks of severe acute respiratory syndrome (SARS) and 1 laboratory-contamination accident. Here we describe a new phylogenetic analytical method to study the sources and dissemination paths of SARS-associated coronavirus (SARS-CoV) infections in Taiwan Methods A phylogenetic analytical tool for combining nucleotide sequences from 6 variable regions of a SARS-CoV genome was developed by use of 20 published SARS-CoV sequences; and this method was validated by use of 80 published SARS-CoV sequences. Subsequently, this new tool was applied to provide a better understanding of the entire complement of Taiwanese SARS-CoV isolates, including 20 previously published and 19 identified in this study. The epidemiological data were integrated with the results from the phylogenetic tree and from the nucleotide-signature pattern Results The topologies of phylogenetic trees generated by the new and the conventional strategies were similar, with the former having better robustness than the latter, especially in comparison with the maximum-likelihood trees: the new strategy revealed that during 2003 there were 5 waves of epidemic SARS-CoV infection, which belonged to 3 phylogenetic clusters in Taiwan Conclusions The new strategy is more efficient than its conventional counterparts. The outbreaks of SARS in Taiwan originated from multiple sources

negative: Discrete Wavelet Packet Transform Based Discriminant Analysis for Whole Genome Sequences. In recent years, alignment-free methods have been widely applied in comparing genome sequences, as these methods compute efficiently and provide desirable phylogenetic analysis results. These methods have been successfully combined with hierarchical clustering methods for finding phylogenetic trees. However, it may not be suitable to apply these alignment-free methods directly to existing statistical classification methods, because an appropriate statistical classification theory for integrating with the alignment-free representation methods is still lacking. In this article, we propose a discriminant analysis method which uses the discrete wavelet packet transform to classify whole genome sequences. The proposed alignment-free representation statistics of features follow a joint normal distribution asymptotically. The data analysis results indicate that the proposed method provides satisfactory classification results in real time.

negative: The E Protein Is a Multifunctional Membrane Protein of SARS-CoV The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH(2)-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

negative: Composition of human-specific slow codons and slow di-codons in SARS-CoV and 2019-nCoV are lower than other coronaviruses suggesting a faster protein synthesis rate of SARS-CoV and 2019-nCoV Translation of a genetic codon without a cognate tRNA gene is affected by both the cognate tRNA availability and the interaction with non-cognate isoacceptor tRNAs. Moreover, two consecutive slow codons (slow di-codons) lead to a much slower translation rate. Calculating the composition of host specific slow codons and slow di-codons in the viral protein coding sequences can predict the order of viral protein synthesis rates between different virus strains. Comparison of human-specific slow codon and slow di-codon compositions in the genomes of 590 coronaviruses infect humans revealed that the protein synthetic rates of 2019 novel coronavirus (2019-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV) may be much faster than other coronaviruses infect humans. Analysis of host-specific slow codon and di-codon compositions provides links between viral genomic sequences and capability of virus replication in host cells that may be useful for surveillance of the transmission potential of novel viruses.

negative: ACE2 polymorphisms and individual susceptibility to SARS-CoV-2 infection: insights from an in silico study The current SARS covid-19 epidemic spread appears to be influenced by ethnical, geographical and sex-related factors that may involve genetic susceptibility to diseases. Similar to SARS-CoV, SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells, notably type II alveolar epithelial cells. Importantly, ACE2 gene is highly polymorphic. Here we have used in silico tools to analyze the possible impact of ACE2 single-nucleotide polymorphisms (SNPs) on the interaction with SARS-CoV-2 spike glycoprotein. We found that S19P (common in African people) and K26R (common in European people) were, among the most diffused SNPs worldwide, the only two SNPs that were able to potentially affect the interaction of ACE2 with SARS-CoV-2 spike. FireDock simulations demonstrated that while S19P may decrease, K26R might increase the ACE2 affinity for SARS-CoV-2 Spike. This finding suggests that the S19P may genetically protect, and K26R may predispose to more severe SARS-CoV-2 disease.

negative: Unfolding SARS-CoV-2 viral genome to understand its gene expression regulation SARS-CoV-2 is a new virus responsible for an outbreak of respiratory illness known as COVID-19, which has spread to several countries around the world and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. In silico analysis of the transcription start sites, promoter regions, transcription factors and their binding sites, gene ontology, CpG islands for SARS-CoV-2 viral genome are a first step to understand the regulation mechanisms of gene expression and association with genetic variations in the genomes. For this purpose, we first computationally surveyed all SARS-CoV-2 virus genes with the open reading frames from NCBI database and found eleven sequences to accomplish the mentioned features by using bioinformatics tools. Our analysis revealed that all (100%) of the SARS-CoV-2 virus genes have more than one TSS. By taking all TSSs with the highest predictive score we determined promoter regions and identified five common candidate motifs (MVI, MVII, MVIII, MVIV and MVV) of which MVI was found to be shared by all promoter regions of SARS-CoV-2 virus genes with the least E-value (3.8e-056, statistically highly significant). In our further analysis of MVI we showed MVI serve as binding sites for a single transcription factor (TF) family, EXPREG, involved in the regulatory mode of these genes. From EXPREG family four TFs that belongs to Cyclic AMP (cAMP) receptor protein (CRP) and Catabolite control protein A (CcpA) group mostly serve as transcriptional activator whereas two TFs that belong to LexA group always serve as transcriptional repressor in different kinds of cellular processes and molecular functions. Therefore, we unfolded SARS-CoV-2 viral genome to shed light on its gene expression regulation that could help to design and evaluate diagnostic tests, to track and trace the ongoing outbreak and to identify potential intervention options.

negative: Phylogenetics Algorithms and Applications Phylogenetics is a powerful approach in finding evolution of current day species. By studying phylogenetic trees, scientists gain a better understanding of how species have evolved while explaining the similarities and differences among species. The phylogenetic study can help in analysing the evolution and the similarities among diseases and viruses, and further help in prescribing their vaccines against them. This paper explores computational solutions for building phylogeny of species along with highlighting benefits of alignment-free methods of phylogenetics. The paper has also discussed the application of phylogenetic study in disease diagnosis and evolution.

negative: Guillain-Barré syndrome and SARS-CoV-2 Since January 2020, after Chinese health authorities identified a new type of coronavirus (SARS-CoV-2), the virus has spread throughout China and consecutively throughout the whole world. The most common symptoms include fever and respiratory tract symptoms. Nevertheless, some patients show less common symptoms such as gastrointestinal or neurological manifestations. This article presents the case of a 65-years old man who was presumptively infected with SARS-CoV-2 during his ski vacation in Austria in March 2020 and acutely presented with typical symptoms of Guillain-Barré syndrome.

negative: Genomics functional analysis and drug screening of SARS-CoV-2 Abstract A novel coronavirus appeared in Wuhan, China has led to major outbreaks. Recently, rapid classification of virus species, analysis of genome and screening for effective drugs are the most important tasks. In the present study, through literature review, sequence alignment, ORF identification, motif recognition, secondary and tertiary structure prediction, the whole genome of SARS-CoV-2 were comprehensively analyzed. To find effective drugs, the parameters of binding sites were calculated by SeeSAR. In addition, potential miRNAs were predicted according to RNA base-pairing. After prediction by using NCBI, WebMGA and GeneMark and comparison, a total of 8 credible ORFs were detected. Even the whole genome have great difference with other CoVs, each ORF has high homology with SARS-CoVs (>90%). Furthermore, domain composition in each ORFs was also similar to SARS. In the DrugBank database, only 7 potential drugs were screened based on the sequence search module. Further predicted binding sites between drug and ORFs revealed that 2-(N-Morpholino)-ethanesulfonic acid could bind 1# ORF in 4 different regions ideally. Meanwhile, both benzyl (2-oxopropyl) carbamate and 4-(dimehylamina) benzoic acid have bene demonstrated to inhibit SARS-CoV infection effectively. Interestingly, 2 miRNAs (miR-1307-3p and miR-3613-5p) were predicted to prevent virus replication via targeting 3′-UTR of the genome or as biomarkers. In conclusion, the novel coronavirus may have consanguinity with SARS. Drugs used to treat SARS may also be effective against the novel virus. In addition, altering miRNA expression may become a potential therapeutic schedule.

negative: Genetic Analysis Genetic analysis of sequence data is central to determining the evolutionary history and molecular epidemiology of viruses, particularly those such as influenza A virus that have complex ecosystems involving multiple hosts. Here we provide an outline of routine phylogenetic analyses of influenza A viruses including multiple sequence alignment, selecting the best-fit evolutionary model and phylogenetic tree reconstruction using Neighbor joining, Maximum likelihood, and Bayesian inference.

negative: Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin Abstract Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl 6 , ( C57Bl 6 × A J )F1 , and A J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl 6 and ( C57Bl 6 × A J )F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl 6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl 6 mice. A single dose of dieldrin did not alter the in vivo resistance of A J animals to acute MHV3 disease. The resistant A J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl 6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl 6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl 6 mice.

negative: Limited SARS-CoV-2 diversity within hosts and following passage in cell culture Since the first reports of pneumonia associated with a novel coronavirus (COVID-19) emerged in Wuhan, Hubei province, China, there have been considerable efforts to sequence the causative virus, SARS-CoV-2 (also referred to as hCoV-19) and to make viral genomic information available quickly on shared repositories. As of 30 March 2020, 7,680 consensus sequences have been shared on GISAID, the principal repository for SARS-CoV-2 genetic information. These sequences are primarily consensus sequences from clinical and passaged samples, but few reports have looked at diversity of virus populations within individual hosts or cultures. Understanding such diversity is essential to understanding viral evolutionary dynamics. Here, we characterize within-host viral diversity from a primary isolate and passaged samples, all originally deriving from an individual returning from Wuhan, China, who was diagnosed with COVID-19 and subsequently sampled in Wisconsin, United States. We use a metagenomic approach with Oxford Nanopore Technologies (ONT) GridION in combination with Illumina MiSeq to capture minor within-host frequency variants ≥1%. In a clinical swab obtained from the day of hospital presentation, we identify 15 single nucleotide variants (SNVs) ≥1% frequency, primarily located in the largest gene – ORF1a. While viral diversity is low overall, the dominant genetic signatures are likely secondary to population size changes, with some evidence for mild purifying selection throughout the genome. We see little to no evidence for positive selection or ongoing adaptation of SARS-CoV-2 within cell culture or in the primary isolate evaluated in this study. Author Summary Within-host variants are critical for addressing molecular evolution questions, identifying selective pressures imposed by vaccine-induced immunity and antiviral therapeutics, and characterizing interhost dynamics, including the stringency and character of transmission bottlenecks. Here, we sequenced SARS-CoV-2 viruses isolated from a human host and from cell culture on three distinct Vero cell lines using Illumina and ONT technologies. We show that SARS-CoV-2 consensus sequences can remain stable through at least two serial passages on Vero 76 cells, suggesting SARS-CoV-2 can be propagated in cell culture in preparation for in-vitro and in-vivo studies without dramatic alterations of its genotype. However, we emphasize the need to deep-sequence viral stocks prior to use in experiments to characterize sub-consensus diversity that may alter outcomes.

negative: Pulmonary embolism in COVID-19. When nothing is what it seems

negative: Computational analysis suggests putative intermediate animal hosts of the SARS-CoV-2 The authors have withdrawn their manuscript whilst they wish to perform additional experiments to validate their conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author for more details.

negative: Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak Abstract Objectives In this study, five SARS-CoV-2 PCR assay panels were evaluated against the accumulated genetic variability of the virus to assess the effect on sensitivity of the individual assays. Design or methods As of week 21, 2020, the complete set of available SARS-CoV-2 genomes from GISAID and GenBank databases were used in this study. SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated. Results A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay. Variations in 8,773 (25.94%) genomes were considered as high risk, whereas additional 2,854 (8.43%) genomes presented low frequent single mutations and were predicted to yield no impact on sensitivity. In case of the QIAstat-Dx SARS-CoV-2 Panel, 99.11% of the genomes matched with a 100% coverage all oligonucleotides, and critical variations were tested in vitro corroborating no loss of sensitivity. Conclusions This analysis stresses the importance of targeting more than one region in the viral genome for SARS-CoV-2 detection to mitigate the risk of loss of sensitivity due to the unknown mutation rate during this SARS-CoV-2 outbreak.INTRODUCTIONSince the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 (Zhu et al., 2020), the number of confirmed cases has been rising dramatically (The Open Science Prize, 2020). According to data from the World Health Organization (WHO), Coronavirus Disease 2019 or COVID-19 (Gorbalenya et al., 2020) has been diagnosed in over 6,057,853 people from more than 200 countries or territories and caused over 371,166 deaths worldwide as of 1 June 2020 (World Health Organization, 2020a).SARS-CoV-2 is the seventh coronavirus known to infect humans and it appears to have its origin from zoonotic transmission similar to SARS-CoV (2002) and MERS-CoV (2012) (Andersen et al., 2020). SARS-CoV-2 is hypothesised to be the product of recombination or mutation from a genetically related SARS-CoV species hosted by bats (Sun et al., 2020). Unlike seasonal human coronaviruses HKU1, NL63, OC43 and 229E which are associated with mild symptoms (Corman et al., 2018), SARS-CoV-2 can cause pneumonia and severe acute respiratory syndrome (Andersen et al., 2020). All coronaviruses are single, positive-stranded RNA viruses with similar genomic ∼30kb size and structure. These genes encode non-structural proteins (ORF1a and ORF1b) involved in replication at the 5′-end and multiple structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) downstream that make up the virus particle (Chan et al., 2020; Wu et al., 2020).During novel RNA viral outbreaks, detection of the virus relies on real-time reverse transcription polymerase chain reaction (real-time RT-PCR) for detection of the RNA of the virus In the case of SARS-CoV-2, RT-PCR is used for confirmatory diagnosis of symptomatic patients as well as increasingly screening of asymptomatic contacts and subjects at risk. The unprecedented outbreak posed the challenge of evaluating the different diagnostic RT-PCR assays sensitivity and specificity without a previous established gold standard.Since the availability of the first SARS-CoV-2 genomic sequence (Wu et al., 2020), several studies have reported a rapid genetic evolution of SARS-CoV-2 through a phylogenetic tree tracking the geographical spread of the virus (Andersen et al., 2020; The Open Science Prize, 2020; Wu et al., 2020; Yi, 2020). As previously established for RNA viruses, the nucleotide mutation rate of SARS-CoV-2 is estimated to be 8E-04 substitutions per site per year as a consequence of the lack of proofreading activity of polymerases (Lauring and Andino, 2020; The Open Science Prize, 2020). It evinces that new genetic variations of SARS-CoV-2 that will most likely occur during this evolving outbreak could compromise the sensitivity and specificity of RT-PCR detection (Lippi et al., 2020).Specifically, both natural mutation rate of a virus (Lauring and Andino, 2020) and active viral recombination (Yi, 2020) could potentially impair the efficiency of oligonucleotide annealing (Lippi et al., 2020), affecting sensitivity (increasing the limit of detection) or inclusivity (missed detection of some strains due to genomic changes). We hypothesize that in order to minimize the risk of sensitivity loss of RT-PCR for detecting novel viruses with unknown genetic variability like SARS-CoV-2, published RT-PCR design strategies for SARS-CoV-2 detection should target more than one region in the viral genome. This was the general approach followed to obtain the earliest available SARS-CoV-2 RT-PCR assays in panels used worldwide (Centers for Disease Control and Prevention, 2020a, 2020b; Chu et al., 2020; Corman et al., 2020; National Microbiology Data Center, 2020; QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020; University of Hong Kong, 2020; World Health Organization, 2020b), including the syndromic multiplex QIAstat-Dx Respiratory SARS-CoV-2 Panel (Table 1). The selected conserved target regions from available SARS-CoV-2 genomes minimizes the potential cross-reactivity with other coronaviruses (Gorbalenya et al., 2020; Zhu et al., 2020), including human coronaviruses (Wu et al., 2020) and bat-SARS-like coronaviruses (Chan et al., 2020).The goal of this study is to assess how the genetic variability observed in the SARS-CoV-2 genome as of week 21, 2020 has affected sensitivity of publicly available SARS-CoV-2 real-time RT-PCR Panel assays. Additionally, a detailed characterization and evaluation of the impact on performance of the SARS-CoV-2 assays in the QIAstat-Dx Respiratory SARS-CoV-2 Panel was carried out.METHODSThe complete set of available SARS-CoV-2 genomes larger than 26,000 nucleotides from GISAID (https://www.gisaid.org/) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) databases were used in this study. As of week 21, 2020, a total of 30,090 and 3,729 available genomes (GISAID and GenBank respectively, Table S1) were aligned using ClustalW algorithm implemented in Geneious software v.10.0.3. (http://www.geneious.com), with a gap open and extent cost of 5 and 3 respectively. The output alignment was curated manually.SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx (Table 1) were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated (Table S2). Variations were considered as high risk when they were placed in the three last nucleotides of the 3’-end, affecting the PCR elongation step (Stadhouders et al., 2010; Whiley and Sloots, 2005) or in the 5’-end of the probe affecting the natural 5’- exonuclease activity of the polymerases (Smith et al., 2002). In addition, were also considered as high risk those variations with a significant presence (>1%) among all published genomes that could become more extended worldwide in the current or potential future spread episodes.RESULTSResults showed large nucleotide insertions (EPI_ISL_416672, EPI_ISL_418061, EPI_ISL_418066, EPI_ISL_416720, EPI_ISL_423027, EPI_ISL_424274, EPI_ISL_424327, EPI_ISL_426134, EPI_ISL_427267, EPI_ISL_429646, EPI_ISL_430896, EPI_ISL_434554, EPI_ISL_437867, EPI_ISL_445217) or fragment gene inversions (EPI_ISL_427289, EPI_ISL_427291) mainly in the polyprotein Orf1ab gene, typically indicating genetic recombination common in coronavirus genomes (Sun et al., 2020; Yi, 2020). Since none of them were placed in binding regions of any of the PCR assays, recombination analysis was considered out of the scope of this current study.A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay. Variations in 8,773 (25.94%) genomes were considered as high risk: a) a trinucleotide variation (GGG to AAC) present in 8,251 of the 8,257 (24.41%) genomes affecting the binding of the N gene NMDC assay forward primer; b) different single variations in the last three positions of the 3’-end or 5’-end (primers and probes respectively)..Genetic variations affecting annealing of more than one oligonucleotide of the same assay were not frequent: a) a total of 126 out of the 128 genomes with a non-critical mismatch in Reverse primer of the WHO RdRp assay (Table 2) present any other variation in the Probe (RdRp_SARSr-P2) or in the Forward primer binding regions, b) 17 out of the 8,251 genomes with multiple three consecutive mismatches at the 5’-end (GGG to AAC) of the NMDC N assay presented additional single variations in the Probe or Reverse primer binding regions; c) five genome sequences (EPI_ISL_444736 for the WHO N assay; EPI_ISL_437536, EPI_ISL_422983 and MT293178 for the CDC N1 assay; and EPI_ISL_446918 for the CDC N2 assay) presented combinations of non-critical mismatches among different oligonucleotides of the same set.Additional 2,854 (8.44%) genomes presented low frequent single mutations considered to be low risk (Table 2) and were predicted to yield no impact on sensitivity. However, the effect in every PCR performance must be evaluated individually based on the corresponding PCR thermal profile conditions. The impact of non-3 terminal mismatches should not be underestimated, especially in a diagnostic environment (Stadhouders et al., 2010; Whiley and Sloots, 2005).A large number of genomes with variations affecting the CDC N3 assay were also found (546 variations, 1.61%). Although they cannot be classified as critical based on the parameters defined in the present study, this data supports why the initial N3 CDC assay was removed from the official panel due to inconclusive results (Centers of Disease Control and Prevention, 2020a, 2020b).This same method of evaluation was carried out with the QIAstat-Dx SARS-CoV-2 Panel oligonucleotide sequences. The SARS-CoV-2 assay was originally developed as a real-time RT-PCR duplex assay covering two highly conserved target genes (RdRp and E) reported with the same fluorophore and showing a verified combined limit of detection (LoD) of 500 copies/mL in clinical samples (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020). A total of 33,518 (99.11 %) of 33,819 matched with a 100% coverage all oligonucleotides included in the SARS-CoV-2 assays of the QIAstat-Dx Respiratory SARS-CoV-2 Panel. In contrast, 301 (0.89%) genomes showed a single variation affecting annealing for any oligonucleotide, and 35 (0.10%) of those 301 genomes contained a variation in any critical position of any primer or probe end. Among these latter ones, the most frequent variation consisted of a mismatch in the last position of the 3’-end of one of the primers (7 genomes, 0.02%). Because a mismatch in this position might impair the elongation PCR step (Stadhouders et al., 2010; Whiley and Sloots, 2005), this genomic mutation was further tested in vitro re-evaluating the LoD. A total of twenty replicates of quantified synthetic dsDNA reproducing the sequence containing this mutation were run at the LoD concentration (500 copies/mL) described in the publicly available QIAstat-Dx Respiratory SARS-CoV-2 Panel instructions for use (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020), and 19 out of the 20 (95%) replicates were positive (Table S3) confirming the defined LoD. Therefore, the QIAstat-Dx SARS-CoV-2 assay remains highly specific and sensitive against SARS-CoV-2 even in light of genomic variations from the first three months of the COVID-19 outbreak (Visseaux et al., 2020).DISCUSSIONGenetic variability observed among SARS-CoV-2 genomes available until week 15, 2020 after the onset of the outbreak showed single nucleotide mutations affecting the annealing of all SARS-CoV-2 RT-PCR panels evaluated in this study. The majority of the annotated single nucleotide variations are predicted to have no effect on sensitivity at panel level, including those placed in critical positions. This was confirmed for the QIAstat-Dx SARS-CoV-2 Panel to show no effect on the LoD of the panel.Given that genetic variability in the SARS-CoV-2 genome is expected to increase based on the natural viral mutation and recombination rates, our results show that combination of more than one assay target in real-time RT-PCR SARS-CoV-2 panels can mitigate the risk of loss of sensitivity or specificity. In this regards, continuous monitoring of genomic variations is essential to provide a rapid response in case assay re-design is needed.AcknowledgementsWe thank all those who have contributed sequences to the GISAID database (https://www.gisaid.org/) and to NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). We also thank the QIAstat-Dx Barcelona team for their support in their corresponding roles.Conflict of interests: LP, RP, MR, SNR, MJF,DM,MLF and JP are QIAGEN employees.No ethical approval was required to conduct this work. The work described in this paper was funded by the employer of the authors (QIAGEN).ReferencesAndersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med 2020. https://doi.org/10.1038/s41591-020-0820-9.Centers for Disease Control and Prevention (CDC). 2019-novel coronavirus (2019-nCoV) real-time rRT-PCR panel primers and probes. 2020a; Accessed: 09 Apr 2020. Available from: https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf.Centers for Disease Control and Prevention (CDC). CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel. 2020b; Accessed: 09 Apr 2020. Available from: https://www.fda.gov/media/134922/download.Chan JFW, Kok KH, Zhu Z, Chu H, To KKW, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 2020; 9: 221-36. https://doi.org/10.1080/22221751.2020.1719902.Chu DKV, Pan Y, Cheng SMS, Hui KPY, Krishnan P, Liu Y, et al. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin Chem 2020; 0(0):1-7. https://doi.org/10.1093/clinchem/hvaa029.Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by realtimeRT-PCR. Euro Surveill 2020; 25(3): pii=2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045.Corman VM, Muth D, Niemeyer D, Drosten C. Hosts and sources of endemic human coronaviruses. Adv Virus Res 2018; 100:163-188. ISSN 0065-3527. https://doi.org/10.1016/bs.aivir.2018.01.001.Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. 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The effects of sequence length and oligonucleotide mismatches on 5’ exonuclease assay efficiency. Nucleic Acids Res. 2002; 30(20): e111. https://doi.org/10.1093/nar/gnf110.Stadhouders R, Pas SD, Anber J, Voermans J, Mes THM, Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5’ nuclease assay. J Mol Diagn 2010; 12(1): 109-117. https://doi.org/10.2353/jmoldx.2010.090035.Sun J, He WT, Wang L, Lai A, Ji X, Zhai X et al. COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives. Trends Mol. Med0 2020; 1550. https://doi.org/10.1016/j.molmed.2020.02.008.The Open Science Prize. Nextstrain: Real-time tracking of pathogen evolution. Genomic epidemiology of novel coronavirus. 2020; Accessed: 09 Apr 2020. Available from: https://nextstrain.org/ncov.The University of Hong Kong (HKU), School of Public Health. Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR. 2020; Accessed: 09 Apr 2020. Available from: https://www.who.int/docs/default-source/coronaviruse/peiris-protocol-16-1-20.pdf?sfvrsn=af1aac73_4.Visseaux B, Le Hingrat Q, Collin G, Bouzid D, Lebourgeois S, Le Pluart D et al. Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV-2 detection. J Clin Microbiol 2020;JCM.00630-20. https//doi.org/10.1128/JCM.00630-20.Whiley DM and Sloots TP. Sequence variation in primer targets affects the accuracy of viral quantitative PCR. J Clin Virol 2005; 34: 104–107. https://doi.org/10.1016/j.jcv.2005.02.010.World Health Organization (WHO). Coronavirus disease 2019 (COVID-19) situation report -79. 2020a; Accessed: 09 Apr 2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/.World Health Organization (WHO). Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases. 2020b; Accessed: 09 Apr 2020. Available from: https://apps.who.int/iris/rest/bitstreams/1271387/retrieve.Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature 2020; 579: 265–269. https://doi.org/10.1038/s41586-020-2008-3.Yi H. 2019 novel coronavirus is undergoing active recombination. Clin Infect Dis 2020; ciaa219. https://doi.org/10.1093/cid/ciaa219.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 2020; 382(8): 727-33. https://doi.org/10.1056/NEJMoa2001017.Authors contributionsLP: Planned experiments, conceptualised the laboratory work, conceptualised the overall study, wrote the manuscript draft.MR: Planned and conducted experiments.RP: Planned and conducted experiments.SR: Contributed to overall study planning and review of the manuscript.MJ: Contributed to overall study planning and review of the manuscript.DM: Contributed to overall study conceptualization and review of the manuscript.MLF: Contributed to overall study planning and review of the manuscript.JP: Contributed to overall study conceptualization, co-wrote of the manuscript.Table 1. Description of earliest available SARS-CoV-2 RT-PCR assays RT-qPCR panel Assays included Target genes Description f References WHO panel a 3RdRp, E, Nthe E gene assay is used as the firstline screening tool, then followed by confirmatory testing with an RdRp gene assay. The N gene assay can eventually be analyzed as an additional confirmatory assay.Corman et al., 2020; World Health Organization, 2020bCDC (US) panel b 2NThis panel contains two monoplex assays (N1, N2) designed for specific detection of SARS-CoV-2. A specimen is considered positive for 2019-nCoV if both assays are positive, whereas if only one assay resulted positive the result is inconclusive and it should be retested.Centers of Disease Control and Prevention, 2020a, 2020bNMDC panel c 2RdRp, NThis panel contains two monoplex assays designed for specific detection of SARS-CoV-2. A specimen is considered positive for 2019-nCoV if both assays are positive.National Microbiology Data Center, 2020HKU panel d 2RdRp, NThe N gene RT-PCR is recommended as a screening assay and the Orf1b assay as a confirmatory one (the N gene assay is about 10 times more sensitive than the ORF-1b gene assay in detecting positive clinical specimens).Chu et al., 2020; The University of Hong Kong, 2020.QIAstat-Dx Panel e 2RdRp, EThis panel contains a Duplex assay in the same optical channel to report an additive performance (including CT and endpoint fluorescence values) of the two PCR assays and it discriminates SARS-CoV-2 detection from more than other 20 viruses and bacteria responsible for upper respiratory infections including other endemic coronavirusesQIAstat-Dx Respiratoyry SARS-CoV-2 Panel, 2020RdRp: RNA-dependent RNA polymerase gene (inside the Orf1ab polyprotein gene), E: envelop gene, N: Nucleocaspide gene.This panel was originally proposed by the Charité-Universitätsmedizin Berlin Institute of Virology (Corman et al., 2020), and then endorsed by the WHO (World Helath Organization, 2020b).Initial CDC panel contained one additional assay (N3) designed for universal detection of SARS-like coronaviruses (Centers of Disease Control and Prevention, 2020a) but has been removed due to inconclusive results based on low performance (Centers of Disease Control and Prevention, 2020b).National Microbiology Data Center (NMDC) works in collaboration with National Institute for Viral Disease Control and Prevention under Chinese Center for Disease Control and Prevention.The School of Public Health of the University of Hong Kong (HKU) developed the assay (Chu et al., 2020) and it was later established as official protocol under the WHO Network Laboratories (The University of Hong Kong, 2020).QIAstat-Dx Panel is a syndromic Point-of-Care system incorporating sample purification and real time RT-qPCR amplification for more than 20 viral and bacterial respiratory pathogens, including SARS-CoV-2.WHO and HKU assays are reactive with coronaviruses under the subgenus Sarbecovirus that includes 2019-nCoV, SARS-CoV and bat SARS-like coronaviruses. Since SARS was eliminated in humans since 2004, individuals with samples that are positive in these RT-PCR assays should be infected by the 2019-nCoV or its related animal coronaviruses.Table 2. Summary of genomic variants detected in complementary binding regions of earliest available SARS-CoV-2 RT-PCR assays. RT-qPCR panel assay Type Oligonucleotide name Number of genomes (%) with non-critical single variations Number of genomes (%) with high risk single variations WHO panelEFwE_Sarbeco_F10 (0.030%)2 (0.006%)aPE_Sarbeco_P134 (0.101%)9 (0.027%)bRevE_Sarbeco_R14 (0.4041%)-RdRpFwRdRp_SARSr-F95 (0.281%)5 (0.015%)cPRdRp_SARSr-P1126 (0.373%)2 (0.006%)dPRdRp_SARSr-P215 (0.044%)-RevRdRp_SARSr-R128 (0.378%)-NFwN_Sarbeco_F70 (0.207%)4 (0.012%)ePN_Sarbeco_P169 (0.204%)11 (0.033%)fRevN_Sarbeco_R119 (0.352%)1 (0.003%)g CDC (US) panelN1Fw2019-nCoV_N1-F40 (0.118%)3 (0.009%)hP2019-nCoV_N1-P53 (0.157%)353 (1.044%)iRev2019-nCoV_N1-R168 (0.497%)4 (0.012%)jN2Fw2019-nCoV_N2-F58 (0.172%)-P2019-nCoV_N2-P102 (0.302%)41 (0.121%)kRev2019-nCoV_N2-R41 (0.121%)-N3Fw2019-nCoV_N3-F344 (1.017%)l-P2019-nCoV_N3-P107 (0.316%)6 (0.018%)mRev2019-nCoV_N3-R86 (0.254%)3 (0.009%)n NMDC panelRdRpFwForward primer23 (0.068%)-PFluorescent probe48 (0.142%)10 (0.030%)oRevReverse primer30 (0.089%)1 (0.003%)pNFwForward primer337 (0.996%)8257 (24.415%)qPFluorescent probe15 (0.044%)3 (0.009%)rRevReverse primer56 (0.166%)5 (0.015%)s HKU panelRdRpFwHKU-ORF1b-nsp14F84 (0.248%)5 (0.015%)PHKU-ORF1b-nsp14P45 (0.133%)-RevHKU-ORF1b-nsp14R62 (0.183%)2 (0.006%)uNFwHKU-NF73 (0.216%)3 (0.009%)vPHKU-NP53 (0.157%)6 (0.018%)wRevHKU-NR83 (0.245%)2 (0.006%)x QIAstat-Dx PanelRdRpFwForward primer15 (0.044%)1 (0.003%)yPFluorescent probe133 (0.393%)12 (0.035%)zRevReverse primer27 (0.080%)3 (0.009%)aaEFwForward primer57 (0.169%)11 (0.032%)bbPFluorescent probe10 (0.030%)1 (0.003%)ccRevReverse primer24 (0.071%)7 (0.021%)dd TOTALS2,854 (8.434%)8,773 (25,941%)ACUMMULATED TOTAL11,627 (34.380%)Fw: Forward primer, P: Probe, Rev: Reverse primerTwo genome sequences with a mismatch in in the three last positions of the 3’-end (U to C and G to respectively).Nine genomes with a mismatch in the last three positions of the 5’-end (five A to G, and four C to U).Five genomes with a mismatch in the last three positions of the 3’-end (G to A, C to U, or G to U).Two genomes with multiple mismatches in the binding position of this probe (positions 1 (C to U), 7 (G to A), 10 (A to G), 12 (R to C), 21 (U to A) of the 5’-end)).Four genomes with a mismatch in any of the three last positions at the 3’-end (C to U, or U to C).Eleven genomes with a mismatch in position 2 of the 5’-end (C to U).One genome with a mismatch in the first position of the 3’-end (G to C).Three genomes with a mismatch in the second position of the 3’-end (A to C/G).A total of 353 genomes containing a mismatch in any of the last three positions of the 5’-end (C to A/U, C to U/G).Four genomes with a mismatch in the third position of the 3’-end (C to A).A total of 41 genomes with a mismatch in the first position of the 5’-end (A to U).A total of 292 genomes present the same mismatch in position 15 of the 3’-end (U to C).Six genomes with a mismatch in second (Y to G) or third (C to U) positions of the 5’-end.Three genomes with a mismatch in first (G to A) or second (U to A) positions of the 3’-end.Ten genomes with any mismatch in the last three positions of the 5’-end (C to A, C to U, G to A).A single genome with a mismatch in the second position of the 3’-end (G to A).A total of 8251 genomes with multiple three consecutive mismatches in positions 20,21,22 of the 3’-end, corresponding to positions 1, 2, 3 of the 5’-end (GGG to AAC). Six additional genomes with mismatches in any of the three last positions of the 3’-end.Three genomes with a mismatch in the second (U to C) or third (G to U) positions of the 5’-end.Five genomes with a mismatch in the first (G to A) or third (C to A/U) positions of the 3’-end.Five genomes with a mismatch in the second (C to U) or third (C to U) positions of the 3’-end.Two genomes with a mismatch in the second (U to C) or third (C to U) positions of the 3’-end.Three genomes with a mismatch in the third (U to C) position of the 3’-end.Six genomes with a mismatch in the second (C to A) or third (A to G) positions of the 5’-end.Two genomes with a mismatch in the second (U to C) or third (A to G) positions of the 3’-end.One mismatch in the third position of the 3’-end.Twelve genomes with a single mismatch in any of the three last positions of the 5’-end.Three genomes with a mismatch in the second position of the 3’-end.Seven genomes with a mismatch in the last position of the 3’-end, together with four additional genomes with a mismatch in the second or third positions.A single genome with a mismatch in the second position of the 5’-end.Six genomes with a mismatch in third position of the 3’-end, and one additional genome with a double mismatch in position 1

negative: Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea Bats have been widely known as natural reservoir hosts of zoonotic diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused by coronaviruses (CoVs). In the present study, we investigated the whole genomic sequence of a SARS-like bat CoV (16BO133) and found it to be 29,075 nt in length with a 40.9% G+C content. Phylogenetic analysis using amino acid sequences of the ORF 1ab and the spike gene showed that the bat coronavirus strain 16BO133 was grouped with the Beta-CoV lineage B and was closely related to the JTMC15 strain isolated from Rhinolophus ferrumequinum in China. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2). Interestingly, 16BO133 showed complete elimination of ORF8 regions induced by a frame shift of the stop codon in ORF7b. The lowest amino acid identity of 16BO133 was identified at the spike region among various ORFs. The spike region of 16BO133 showed 84.7% and 75.2% amino acid identity with Rf1 (SARS-like bat CoV) and Tor2 (human SARS CoV), respectively. In addition, the S gene of 16BO133 was found to contain the amino acid substitution of two critical residues (N479S and T487 V) associated with human infection. In conclusion, we firstly carried out whole genome characterization of the SARS-like bat coronavirus discovered in the Republic of Korea; however, it presumably has no human infectivity. However, continuous surveillance and genomic characterization of coronaviruses from bats are necessary due to potential risks of human infection induced by genetic mutation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11262-019-01668-w) contains supplementary material, which is available to authorized users.

negative: Emergence and Reemergence of Severe Acute Respiratory Syndrome (SARS) Coronaviruses The positive-strand RNA viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and recently emerged COVID-19 epidemics, demonstrated the transmission capability of the coronaviruses by crossing the species barrier and emergence in humans. The source of coronavirus disease 2019 (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), firstly reported in December 2019 at Wuhan, China. COVID-19 is a kind of viral pneumonia. The outbreak of SARS-CoV-2 (COVID-19) has been reported as the introduction of the third highly pathogenic coronavirus which crossed the species barrier and spread into the human population. Severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) were the first two epidemic viruses, respectively, in the twenty-first century. Introduction of the 2019 novel coronaviruses (2019-nCoV) in human population is a worldwide concern, and this might have generated via RNA recombination among the previous reported coronaviruses. The COVID-19 is spreading in an alarming rate, and till date no vaccine or specific medicines are available in the market. The newly emerged coronavirus COVID-19 is strongly related to SARS-CoV except little dissimilarity. In this chapter, we will discuss about the alterations and variations in antigenicity, structural changes, and RNA recombination which might be responsible for the COVID-19 emergence.

negative: Calculation of Evolutionary Correlation between Individual Genes and Full-Length Genome: A Method Useful for Choosing Phylogenetic Markers for Molecular Epidemiology Individual genes or regions are still commonly used to estimate the phylogenetic relationships among viral isolates. The genomic regions that can faithfully provide assessments consistent with those predicted with full-length genome sequences would be preferable to serve as good candidates of the phylogenetic markers for molecular epidemiological studies of many viruses. Here we employed a statistical method to evaluate the evolutionary relationships between individual viral genes and full-length genomes without tree construction as a way to determine which gene can match the genome well in phylogenetic analyses. This method was performed by calculation of linear correlations between the genetic distance matrices of aligned individual gene sequences and aligned genome sequences. We applied this method to the phylogenetic analyses of porcine circovirus 2 (PCV2), measles virus (MV), hepatitis E virus (HEV) and Japanese encephalitis virus (JEV). Phylogenetic trees were constructed for comparisons and the possible factors affecting the method accuracy were also discussed in the calculations. The results revealed that this method could produce results consistent with those of previous studies about the proper consensus sequences that could be successfully used as phylogenetic markers. And our results also suggested that these evolutionary correlations could provide useful information for identifying genes that could be used effectively to infer the genetic relationships.

negative: The ability of single genes vs full genomes to resolve time and space in outbreak analysis Inexpensive pathogen genome sequencing has had a transformative effect on the field of phylodynamics, where ever increasing volumes of data have promised real-time insight into outbreaks of infectious disease. As well as the sheer volume of pathogen isolates being sequenced, the sequencing of whole pathogen genomes, rather than select loci, has allowed phylogenetic analyses to be carried out at finer time scales, often approaching serial intervals for infections caused by rapidly evolving RNA viruses. Despite its utility, whole genome sequencing of pathogens has not been adopted universally and targeted sequencing of loci is common in some pathogen-specific fields. In this study we aim to highlight the utility of sequencing whole genomes of pathogens by re-analysing a well-characterised collection of Ebola virus sequences in the form of complete viral genomes (~19kb long) or the rapidly evolving glycoprotein (GP, ~2kb long) gene. We quantify changes in phylogenetic, temporal, and spatial inference resolution as a result of this reduction in data and compare these to theoretical expectations. We propose a simple intuitive metric for quantifying temporal resolution, i.e. the time scale over which sequence data might be informative of various processes as a quick back-of-the-envelope calculation of statistical power available to molecular clock analyses.

negative: Prevalence and phylogenetic analysis of porcine diarrhea associated viruses in southern China from 2012 to 2018 BACKGROUND: In China, large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since late 2010. To investigate the prevalence and genetic evolution of diarrhea-associated viruses responsible for the outbreaks, a total of 2987 field diarrheal samples collected from 168 pig farms in five provinces in Southern China during 2012–2018 were tested. RESULTS: Porcine epidemic diarrhea virus (PEDV) was most frequently detected virus with prevalence rates between 50.21 and 62.10% in samples, and 96.43% (162/168) in premises, respectively. Porcine deltacoronavirus (PDCoV) was the second prevalent virus with prevalence rates ranging from 19.62 to 29.19% in samples, and 70.24% (118/168) in premises, respectively. Both transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) were detected at low prevalence rates of < 3% in samples and 10.12% in premises. In this study, we identified a newly emerged swine acute diarrhea syndrome coronavirus (SADS-CoV) in diarrheal samples of piglets from Fujian province in Southern China, and the prevalence rate of SADS-CoV was 10.29% (7/68). Co-infections of these diarrhea-associated viruses were common. The most frequent co-infection was PEDV with PDCoV, with an average detection rate of 12.72% (380/2987, ranging from 8.26–17.33%). Phylogenetic analysis revealed that PEDVs circulating in Southern China during the last 7 years were clustered with the variant strains of PEDV in genotype IIa. The most frequent mutations were present in the collagenase equivalent (COE) and epitope regions of the spike gene of the PEDVs currently circulating in the field. Genetic relationships of PDCoVs were closely related with Chinese strains, other than those present in the USA, South Korea, Thailand and Lao’s public. CONCLUSIONS: The findings of this study indicated that variant PEDV, PDCoV, and SADS-CoV were leading etiologic agents of porcine diarrhea, and either mono-infections or co-infections of pathogenic enteric CoVs were common in pigs in Southern China during 2012–2018. Thus, significant attention should be paid in order to effectively prevent and control porcine viral diarrhea.

negative: The value of early transmission dynamic studies in emerging infectious diseases

negative: Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 x 10(6) copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 x 10(6) copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.

negative: Molecular characterisation of a bovine-like rotavirus detected from a giraffe BACKGROUND: Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf. RESULTS: This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. Detailed sequence analysis of VP4 and VP7 revealed significant identity at the amino acid sequence level to Bovine RV (BoRV). CONCLUSION: This study demonstrates the need for continuous surveillance of RV strains in various animal populations, which will facilitate the identification of rotavirus hosts not previously reported. Furthermore, extending typical epidemiology studies to a broader host range will contribute to the timely identification of new emerging strain types.

negative: Genotyping coronavirus SARS-CoV-2: Methods and implications Abstract The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.

negative: Ionizing air affects influenza virus infectivity and prevents airborne-transmission By the use of a modified ionizer device we describe effective prevention of airborne transmitted influenza A (strain Panama 99) virus infection between animals and inactivation of virus (>97%). Active ionizer prevented 100% (4/4) of guinea pigs from infection. Moreover, the device effectively captured airborne transmitted calicivirus, rotavirus and influenza virus, with recovery rates up to 21% after 40 min in a 19 m(3) room. The ionizer generates negative ions, rendering airborne particles/aerosol droplets negatively charged and electrostatically attracts them to a positively charged collector plate. Trapped viruses are then identified by reverse transcription quantitative real-time PCR. The device enables unique possibilities for rapid and simple removal of virus from air and offers possibilities to simultaneously identify and prevent airborne transmission of viruses.

negative: The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses Emerging coronaviruses (CoV) are constant global public health threats to society. Multiple ongoing clinical trials for vaccines and antivirals against CoVs showcase the availability of medical interventions to both prevent and treat the future emergence of highly pathogenic CoVs in human. However, given the diverse nature of CoVs and our close interactions with wild, domestic and companion animals, the next epidemic zoonotic CoV could resist the existing vaccines and antivirals developed, which are primarily focused on Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS CoV). In late 2019, the novel CoV (SARS-CoV-2) emerged in Wuhan, China, causing global public health concern. In this review, we will summarize the key advancements of current vaccines and antivirals against SARS-CoV and MERS-CoV as well as discuss the challenge and opportunity in the current SARS-CoV-2 crisis. At the end, we advocate the development of a “plug-and-play” platform technologies that could allow quick manufacturing and administration of broad-spectrum countermeasures in an outbreak setting. We will discuss the potential of AAV-based gene therapy technology for in vivo therapeutic antibody delivery to combat SARS-CoV-2 outbreak and the future emergence of severe CoVs.

negative: NeoCoV Is Closer to MERS-CoV than SARS-CoV Recently, Coronavirus has been given considerable attention from the biomedical community based on the emergence and isolation of a deadly coronavirus infecting human. To understand the behavior of the newly emerging MERS-CoV requires knowledge at different levels (epidemiologic, antigenic, and pathogenic), and this knowledge can be generated from the most related viruses. In this study, we aimed to compare between 3 species of Coronavirus, namely Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome (SARS-CoV), and NeoCoV regarding whole genomes and 6 similar proteins (E, M, N, S, ORF1a, and ORF1ab) using different bioinformatics tools to provide a better understanding of the relationship between the 3 viruses at the nucleotide and amino acids levels. All sequences have been retrieved from National Center for Biotechnology Information (NCBI). Regards to target genomes’ phylogenetic analysis showed that MERS and SARS-CoVs were closer to each other compared with NeoCoV, and the last has the longest relative time. We found that all phylogenetic methods in addition to all parameters (physical and chemical properties of amino acids such as the number of amino acid, molecular weight, atomic composition, theoretical pI, and structural formula) indicated that NeoCoV proteins were the most related to MERS-CoV one. All phylogenetic trees (by both maximum-likelihood and neighbor-joining methods) indicated that NeoCoV proteins have less evolutionary changes except for ORF1a by just maximum-likelihood method. Our results indicated high similarity between viral structural proteins which are responsible for viral infectivity; therefore, we expect that NeoCoV sooner may appear in human-related infection.

negative: Supporting pandemic response using genomics and bioinformatics: A case study on the emergent SARS‐CoV‐2 outbreak Pre‐clinical responses to fast‐moving infectious disease outbreaks heavily depend on choosing the best isolates for animal models that inform diagnostics, vaccines and treatments. Current approaches are driven by practical considerations (e.g. first available virus isolate) rather than a detailed analysis of the characteristics of the virus strain chosen, which can lead to animal models that are not representative of the circulating or emerging clusters. Here, we suggest a combination of epidemiological, experimental and bioinformatic considerations when choosing virus strains for animal model generation. We discuss the currently chosen SARS‐CoV‐2 strains for international coronavirus disease (COVID‐19) models in the context of their phylogeny as well as in a novel alignment‐free bioinformatic approach. Unlike phylogenetic trees, which focus on individual shared mutations, this new approach assesses genome‐wide co‐developing functionalities and hence offers a more fluid view of the ‘cloud of variances’ that RNA viruses are prone to accumulate. This joint approach concludes that while the current animal models cover the existing viral strains adequately, there is substantial evolutionary activity that is likely not considered by the current models. Based on insights from the non‐discrete alignment‐free approach and experimental observations, we suggest isolates for future animal models.

negative: Neutralizing Antibodies Isolated by a site-directed Screening have Potent Protection on SARS-CoV-2 Infection Neutralizing antibody is one of the most effective interventions for acute pathogenic infection. Currently, over three million people have been identified for SARS-CoV-2 infection but SARS-CoV-2-specific vaccines and neutralizing antibodies are still lacking. SARS-CoV-2 infects host cells by interacting with angiotensin converting enzyme-2 (ACE2) via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein. Therefore, blocking the interaction of SARS-CoV-2-RBD and ACE2 by antibody would cause a directly neutralizing effect against virus. In the current study, we selected the ACE2 interface of SARS-CoV-2-RBD as the targeting epitope for neutralizing antibody screening. We performed site-directed screening by phage display and finally obtained one IgG antibody (4A3) and several domain antibodies. Among them, 4A3 and three domain antibodies (4A12, 4D5, and 4A10) were identified to act as neutralizing antibodies due to their capabilities to block the interaction between SARS-CoV-2-RBD and ACE2-positive cells. The domain antibody 4A12 was predicted to have the best accessibility to all three ACE2-interfaces on the spike homotrimer. Pseudovirus and authentic SARS-CoV-2 neutralization assays showed that all four antibodies could potently protect host cells from virus infection. Overall, we isolated multiple formats of SARS-CoV-2-neutralizing antibodies via site-directed antibody screening, which could be promising candidate drugs for the prevention and treatment of COVID-19.

negative: Site-specific glycan analysis of the SARS-CoV-2 spike The emergence of the betacoronavirus, SARS-CoV-2, the causative agent of COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

negative: Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has the capability of gaining rapid mutations as the virus spreads. Whole-genome sequencing data offers a wide range of opportunities to study mutation dynamics. The advantage of an increasing amount of whole-genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity, and to investigate the implications of those mutations in protein stability and viral transmission. We have identified frequently mutated residues by aligning ~660 SARS-CoV-2 genomes and validated in 10,000 datasets available in GISAID Nextstrain. We further evaluated the potential of these frequently mutated residues in protein structure stability of spike glycoprotein and their possible functional consequences in other proteins. Among the 11 genes, surface glycoprotein, nucleocapsid, ORF1ab, and ORF8 showed frequent mutations, while envelop, membrane, ORF6, ORF7a and ORF7b showed conservation in terms of amino acid substitutions. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Some of the mutations across different proteins showed co-occurrences, suggesting their structural and/or functional interaction among different SARS-COV-2 proteins, and their involvement in adaptability and viral transmission. Analysis of protein structure stability of surface glycoprotein mutants indicated the viability of specific variants and are more prone to be temporally and spatially distributed across the globe. A similar empirical analysis of other proteins indicated the existence of important functional implications of several variants. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing, and containment of the disease.

negative: Complete dimensional collapse in the continuum limit of a delayed SEIQR network model with separable distributed infectivity We take up a recently proposed compartmental SEIQR model with delays, ignore loss of immunity in the context of a fast pandemic, extend the model to a network structured on infectivity and consider the continuum limit of the same with a simple separable interaction model for the infectivities [Formula: see text] . Numerical simulations show that the evolving dynamics of the network is effectively captured by a single scalar function of time, regardless of the distribution of [Formula: see text] in the population. The continuum limit of the network model allows a simple derivation of the simpler model, which is a single scalar delay differential equation (DDE), wherein the variation in [Formula: see text] appears through an integral closely related to the moment generating function of [Formula: see text] . If the first few moments of u exist, the governing DDE can be expanded in a series that shows a direct correspondence with the original compartmental DDE with a single [Formula: see text] . Even otherwise, the new scalar DDE can be solved using either numerical integration over u at each time step, or with the analytical integral if available in some useful form. Our work provides a new academic example of complete dimensional collapse, ties up an underlying continuum model for a pandemic with a simpler-seeming compartmental model and will hopefully lead to new analysis of continuum models for epidemics.

negative: ACE2 receptor polymorphism: susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged in Chinese people in December 2019 and has currently spread worldwide causing the COVID-19 pandemic with more than 150,000 deaths. In order for a SARS-CoV like virus circulating in wild life for a very long time to infect the index case-patient, a number of conditions must be met, foremost among which is the encounter with humans and the presence in homo sapiens of a cellular receptor allowing the virus to bind. Recently it was shown that the SARS-CoV-2 spike protein, binds to the human angiotensin I converting enzyme 2 (ACE2). This molecule is a peptidase expressed at the surface of lung epithelial cells and other tissues, that regulates the renin-angiotensin-aldosterone system. Humans are not equal with respect to the expression levels of the cellular ACE2. Moreover, ACE2 polymorphisms were recently described in human populations. Here we review the most recent evidence that ACE2 expression and/or polymorphism could influence both the susceptibility of people to SARS-CoV-2 infection and the outcome of the COVID-19 disease. Further exploration of the relationship between the virus, the peptidase function of ACE2 and the levels of angiotensin II in SARS-CoV-2 infected patients should help to better understand the pathophysiology of the disease and the multi-organ failures observed in severe COVID-19 cases, particularly heart failure.

negative: Recombinant vesicular stomatitis virus expressing the spike protein of genotype 2b porcine epidemic diarrhea virus: A platform for vaccine development against emerging epidemic isolates Abstract Emerging porcine epidemic diarrhea viruses (PEDVs) have caused large economic losses since 2010, and G2b is the prevalent globally epidemic genotype. Given the fastidious isolation of emerging PEDV in cell culture and difficulties in retaining the isolate infectivity upon further in vitro passage, highly attenuated recombinant vesicular stomatitis virus (rVSVMT) was used as a vector to express the PEDV spike (S) protein, aiming to develop a subunit vaccine against G2b viruses. An S protein with 19 of its cytoplasmic domain amino acids deleted could be incorporated into VSV particles, generating rVSVMT (VSVMT-SΔ19) with high efficiency. Our results suggest that VSVMT-SΔ19 could effectively induce PEDV-specific immunity in pigs via intramuscular, but not intranasal, immunization. Notably, immunizations of sows with VSV MT-SΔ19 provided protective lactogenic immunity against a virulent G2b PEDV challenge in piglets. Consequently, recombinant VSVMT may be a promising platform for preparing a subunit vaccine against PEDV.

negative: Interspecies transmission and emergence of novel viruses: lessons from bats and birds As exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. These warm-blooded vertebrates display high species biodiversity, roosting and migratory behavior, and a unique adaptive immune system, which are favorable characteristics for asymptomatic shedding, dissemination, and mixing of different viruses for the generation of novel mutant, recombinant, or reassortant RNA viruses. The increased intrusion of humans into wildlife habitats and overcrowding of different wildlife species in wet markets and farms have also facilitated the interspecies transmission between different animal species.

negative: Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3′ end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3′ end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

negative: The New Age of Virus Discovery: Genomic Analysis of a Novel Human Betacoronavirus Isolated from a Fatal Case of Pneumonia A new human betacoronavirus in lineage c, tentatively called HCoV-EMC, was isolated from a patient from the Kingdom of Saudi Arabia who died from acute severe pneumonia and renal failure. The viral RNA has been detected in eight additional cases. Sequencing and bioinformatic analysis of the viral genomic RNA showed that it is a novel virus not previously detected in any other species and that its closest relatives are two Asian bat coronaviruses. HCoV-EMC may represent a sporadic spillover to humans from an unknown animal reservoir. In a recent article, van Boheemen et al. demonstrated how state-of-the-art sequencing technology and bioinformatic analysis can quickly provide critical insight into the viral genome sequence, phylogeny, replication strategy, and potential drug and vaccine targets and generate tools to evaluate the possible epidemic risk associated with this novel human virus.

negative: Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage Abstract The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

negative: Chapter Eight Molecular Basis of Coronavirus Virulence and Vaccine Development Abstract Virus vaccines have to be immunogenic, sufficiently stable, safe, and suitable to induce long-lasting immunity. To meet these requirements, vaccine studies need to provide a comprehensive understanding of (i) the protective roles of antiviral B and T-cell-mediated immune responses, (ii) the complexity and plasticity of major viral antigens, and (iii) virus molecular biology and pathogenesis. There are many types of vaccines including subunit vaccines, whole-inactivated virus, vectored, and live-attenuated virus vaccines, each of which featuring specific advantages and limitations. While nonliving virus vaccines have clear advantages in being safe and stable, they may cause side effects and be less efficacious compared to live-attenuated virus vaccines. In most cases, the latter induce long-lasting immunity but they may require special safety measures to prevent reversion to highly virulent viruses following vaccination. The chapter summarizes the recent progress in the development of coronavirus (CoV) vaccines, focusing on two zoonotic CoVs, the severe acute respiratory syndrome CoV (SARS-CoV), and the Middle East respiratory syndrome CoV, both of which cause deadly disease and epidemics in humans. The development of attenuated virus vaccines to combat infections caused by highly pathogenic CoVs was largely based on the identification and characterization of viral virulence proteins that, for example, interfere with the innate and adaptive immune response or are involved in interactions with specific cell types, such as macrophages, dendritic and epithelial cells, and T lymphocytes, thereby modulating antiviral host responses and viral pathogenesis and potentially resulting in deleterious side effects following vaccination.

negative: Receptor utilization of angiotensin converting enzyme 2 (ACE2) indicates a narrower host range of SARS-CoV-2 than that of SARS-CoV Coronavirus pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, SARS-CoV caused SARS pandemic in 2003 and SARS-CoV-2 caused the COVID-19 pandemic recently. Both viruses have been reported to originate from bats. Thus, direct or indirect interspecies transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the interspecies transmission. Angiotensin converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Meanwhile, pangolin CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acids that may determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin CoV. Our studies promote the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.

negative: The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health — The latest 2019 novel coronavirus outbreak in Wuhan, China

negative: SARS-CoV Infection in a Restaurant from Palm Civet Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003–2004 were a waitress at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages. All 6 palm civets at the restaurant were positive for SARS-associated coronavirus (SARS-CoV). Partial spike (S) gene sequences of SARS-CoV from the 2 patients were identical to 4 of 5 S gene viral sequences from palm civets. Phylogenetic analysis showed that SARS-CoV from palm civets in the restaurant was most closely related to animal isolates. SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population.

negative: Bats are key hosts in the radiation of mammal-associated Bartonella bacteria Bats are notorious reservoirs of several zoonotic diseases and may be uniquely tolerant of infection among mammals. Broad sampling has revealed the importance of bats in the diversification and spread of viruses and eukaryotes to other animal hosts. Vector-borne bacteria of the genus Bartonella are prevalent and diverse in mammals globally and recent surveys have revealed numerous Bartonella lineages in bats. We assembled a sequence database of Bartonella strains, consisting of nine genetic loci from 209 previously characterized lineages and 121 new cultured strains from bats, and used these data to perform the most comprehensive phylogenetic analysis of Bartonella to date. This analysis included estimation of divergence dates using a molecular clock and ancestral reconstruction of host associations and geography. We estimate that Bartonella began infecting mammals 62 million years ago near the Cretaceous-Paleogene boundary. Additionally, the radiation of particular Bartonella clades correlate strongly to the timing of diversification and biogeography of mammalian hosts. Bats were inferred to be the ancestral hosts of all mammal-associated Bartonella and appear to be responsible for the early geographic expansion of the genus. We conclude that bats have had a deep influence on the evolutionary radiation of Bartonella bacteria and their spread to other mammalian orders. These results support a ‘bat seeding’ hypothesis that could explain similar evolutionary patterns in other mammalian parasite taxa. Application of such phylogenetic tools as we have used to other taxa may reveal the general importance of bats in the ancient diversification of mammalian parasites. Significance statement Discovering the evolutionary history of infectious agents in animals is important for understanding the process of host adaptation and the origins of human diseases. To clarify the evolution of the Bartonella genus, which contains important human pathogens, we performed phylogenetic analysis on a broad diversity of Bartonella strains, including novel strains from bats. Our results indicate that Bartonella clades diversified along with their mammal hosts over millions of years. Bats appear to be especially important in the early radiation and geographic dispersal of Bartonella lineages. These patterns are consistent with research indicating a chiropteran origin of important human viruses and eukaryotic parasites, suggesting that bats may play a unique role as historical sources of infections to other hosts.

negative: Cell entry mechanisms of SARS-CoV-2 A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochemical and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.

negative: Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.

negative: Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)(3)-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.

negative: Infectivity, virulence, pathogenicity, host-pathogen interactions of SARS and SARS-CoV-2 in experimental animals: a systematic review The outbreak of the SARS-CoV-2 in mainland China with subsequent human to human transmission worldwide had taken up the shape of a devastating pandemic. The ability of the virus to infect multiple species other than humans has currently been reported in experimental conditions. Non-human primates, felines, ferrets, rodents and host of other animals could previously be infected in experimental conditions with SARS-CoV and recently with SARS-CoV-2, both virus using Angiotensin-converting-enzyme 2 receptor for cellular entry. The variations in sequence homology of ACE2 receptor across species is identified as one of the factors determining virulence and pathogenicity in animals. The infection in experimental animals with SARS-CoV or SARS-CoV-2 on most occasions are asymptomatic, however, the virus could multiply within the respiratory tract and extra-pulmonary organs in most of the species. Here, we discuss about the pathogenicity, transmission, variations in angiotensin-converting-enzyme 2 receptor-binding across species and host pathogen interactions of SARS and SARS-CoV-2 in laboratory animals used in research.

negative: Stability analysis of leishmania epidemic model with harmonic mean type incidence rate We discussed anthroponotic cutaneous leishmania transmission in this article, due to its large effect on the community in the recent years. The mathematical model is developed for anthroponotic cutaneous leishmania transmission, and its qualitative behavior is taken under consideration. The threshold number [Formula: see text] of the model is derived using the next-generation method. In the disease-free case, local and global stability is carried out with the condition that [Formula: see text] will be less than one. The global stability at the disease-free equilibrium point has been derived by utilizing the Castillo–Chavez method. On the other hand, at the endemic equilibrium point, the local and global stability holds with some conditions, and [Formula: see text] is greater than unity. The global stability at the endemic equilibrium point is established with the help of a geometrical approach which is the generalization of Lyapunov theory, by using the third additive compound matrix. The sensitivity analysis of the threshold number with other parameters is also taken into account. Several graphs of important parameters are discussed in the last section.

negative: Evolving sequence mutations in the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) has continued to cause sporadic outbreaks of severe respiratory tract infection over the last 8 years. METHODS: Complete genome sequencing using next-generation sequencing was performed for MERS-CoV isolates from cases that occurred in Riyadh between 2015 and 2019. Phylogenetic analysis and molecular mutational analysis were carried out to investigate disease severity. RESULTS: A total of eight MERS-CoV isolates were subjected to complete genome sequencing. Phylogenetic analysis resulted in the assembly of 7/8 sequences within lineage 3 and one sequence within lineage 4 showing complex genomic recombination. The isolates contained a variety of unique amino acid substitutions in ORF1ab (41), the N protein (10), the S protein (9) and ORF4b (5). CONCLUSION: Our study shows that MERS-CoV is evolving. The emergence of new variants carries the potential for increased virulence and could impose a challenge to the global health system. We recommend the sequencing every new MERS-CoV isolate to observe the changes in the virus and relate them to clinical outcomes.

negative: Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis Many human viral diseases are a consequence of a zoonotic event. Some of the diseases caused by these zoonotic events have affected millions of people around the world, some of which have resulted in high rates of morbidity/mortality in humans. Changes in the viral proteins that function as ligands of the host receptor may promote the spillover between species. The most recent of these zoonotic events that have caused an ongoing epidemic of high magnitude is the Covid-19 epidemics caused by SARS-CoV-2. The aim of this study was to determine the mutation(s) in the sequence of the spike protein of the SARS-CoV-2 that might be favoring human to human transmission. An in silico approach was performed, and changes were detected in the S1 subunit of the receptor-binding domain of spike. The observed changes have significant effect on SARS-CoV-2 spike/ACE2 interaction and produce a reduction in the binding energy, compared to the one of the Bat-CoV to this receptor. The data presented in this study suggest a higher affinity of the SARS-Cov-2 spike protein to the human ACE2 receptor, compared to the one of Bat-CoV spike and ACE2. This could be the cause of the rapid viral spread of SARS-CoV-2 in humans.

negative: Genotyping coronavirus SARS-CoV-2: methods and implications The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.

negative: Structure-based Design, Synthesis and Biological Evaluation of Peptidomimetic SARS-CoV 3CLpro Inhibitors Structure-based design, synthesis and biological evaluation of a series of peptidomimetic severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. These inhibitors were designed and synthesized based upon our X-ray crystal structure of inhibitor 1 bound to SARS-CoV 3CLpro. Incorporation of Boc-Ser as the P4-ligand resulted in enhanced SARS-CoV 3CLpro inhibitory activity. Structural analysis of inhibitor-bound X-ray structure revealed high binding affinity towards the enzyme.

negative: Identification of conserved epitopes in SARS-CoV-2 spike and nucleocapsid protein Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December 2019, is a novel virus that causes a severe acute respiratory disease. The virus spike glycoproteins and nucleocapsid proteins are the main targets for the development of vaccines and antiviral drugs, to control the disease spread. We herein study the structural order-disorder propensity and the rates of evolution of these two proteins to characterize their B cell and T cell epitopes, previously suggested to contribute to immune response caused by SARS-CoV-2 infections. We first analyzed the rates of evolution along the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes. For this purpose, we compared orthologs from seven human coronaviruses: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. We then focus on the local, structural order-disorder propensities of the protein regions where the SARS-CoV-2 epitopes are located. We show that the vast majority of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains and some of them are characterized by low rates of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by high rates of evolution, probably to overcome host immunity. In conclusion, our results provide evidence for conserved epitopes that may help to develop long-lasting, broad-spectrum SARS-CoV-2 vaccines.

negative: Quality control of low-frequency variants in SARS-CoV-2 genomes During the current outbreak of COVID-19, research labs around the globe submit sequences of the local SARS-CoV-2 genomes to the GISAID database to provide a comprehensive analysis of the variability and spread of the virus during the outbreak. We explored the variations in the submitted genomes and found a significant number of variants that can be seen only in one submission (singletons). While it is not completely clear whether these variants are erroneous or not, these variants show lower transition/transversion ratio. These singleton variants may influence the estimations of the viral mutation rate and tree topology. We suggest that genomes with multiple singletons even marked as high-covered should be considered with caution. We also provide a simple script for checking variant frequency against the database before submission.

negative: SARS Coronavirus Pathogenesis and Therapeutic Treatment Design Emerging pathogens are either new or newly recognized or those that are increasing in incidence and spread. Since the identity of emerging pathogens from animal reservoirs is difficult to predict, the development for pathogen-specific therapeutics and vaccines is problematic. The highly pathogenic SARS coronavirus (SARS-CoV) emerged from zoonotic pools in 2002 to cause a global epidemic of severe acute respiratory syndrome (SARS). Many patients with SARS-CoV experienced an exacerbated form of disease called acute respiratory distress syndrome (ARDS) requiring mechanical ventilation and supplemental oxygen and half of these patients died. Similar to other viral pathogens like influenza and West Nile Virus, the severity of SARS-CoV disease increased with age. Unfortunately, successful vaccination in the most vulnerable populations is a difficult task because of immunological deficiencies associated with aging (immune senescence). Due to the rapidity of virus emergence, technologies like synthetic biology can be harnessed to facilitate rapid recombinant virus construction for studying the novel virus biology, pathogenesis and the evaluation of therapeutic interventions. Since predicting the antigenic identity of future emergence is difficult, candidate vaccines and therapeutics should have a maximal breadth of cross-protection, and panels of antigenically divergent synthetically reconstructed viruses can be used as tools for this evaluation. We discuss how synthetic reconstruction of many animal and human SARS-CoV has provided a model to study the molecular mechanisms governing emergence and pathogenesis of viral diseases. In addition, we review the evolution, epidemiology, and pathogenesis of epidemic and zoonotic SARS-CoV with focus on the development of broadly reactive therapeutics and vaccines that protect aged populations from the zoonotic pool.

negative: COVID-19 outbreak: less stethoscope, more ultrasound

negative: Dinucleotide repeats in coronavirus SARS-CoV-2 genome: evolutionary implications The ongoing global pandemic of infection disease COVID-19 caused by the 2019 novel coronavirus (SARS-COV-2, formerly 2019-nCoV) presents critical threats to public health and the economy since it was identified in China, December 2019. The genome of SARS-CoV-2 had been sequenced and structurally annotated, yet little is known of the intrinsic organization and evolution of the genome. To this end, we present a mathematical method for the genomic spectrum, a kind of barcode, of SARS-CoV-2 and common human coronaviruses. The genomic spectrum is constructed according to the periodic distributions of nucleotides, and therefore reflects the unique characteristics of the genome. The results demonstrate that coronavirus SARS-CoV-2 exhibits dinucleotide TT islands in the non-structural proteins 3, 4, 5, and 6. Further analysis of the dinucleotide regions suggests that the dinucleotide repeats are increased during evolution and may confer the evolutionary fitness of the virus. The special dinucleotide regions in the SARS-CoV-2 genome identified in this study may become diagnostic and pharmaceutical targets in monitoring and curing the COVID-19 disease.

negative: Recognition and analysis of protein-coding genes in severe acute respiratory syndrome associated coronavirus Motivation: The recent outbreak of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) has necessitated an in-depth molecular understanding of the virus to identify new drug targets. The availability of complete genome sequence of several strains of SARS virus provides the possibility of identification of protein-coding genes and defining their functions. Computational approach to identify protein-coding genes and their putative functions will help in designing experimental protocols. Results: In this paper, a novel analysis of SARS genome using gene prediction method GeneDecipher developed in our laboratory has been presented. Each of the 18 newly sequenced SARS-CoV genomes has been analyzed using GeneDecipher. In addition to polyprotein 1ab(1), polyprotein 1a and the four genes coding for major structural proteins spike (S), small envelope (E), membrane (M) and nucleocapsid (N), six to eight additional proteins have been predicted depending upon the strain analyzed. Their lengths range between 61 and 274 amino acids. Our method also suggests that polyprotein 1ab, polyprotein 1a, S, M and N are proteins of viral origin and others are of prokaryotic. Putative functions of all predicted protein-coding genes have been suggested using conserved peptides present in their open reading frames. Availability: Detailed results of GeneDecipher analysis of all the 18 strains of SARS-CoV genomes are available at http://www.igib.res.in/sarsanalysis.html

negative: One-shot screening of potential peptide ligands on HR1 domain in COVID-19 glycosylated spike (S) protein with deep siamese network The novel coronavirus (2019-nCoV) has been declared to be a new international health emergence and no specific drug has been yet identified. Several methods are currently being evaluated such as protease and glycosylated spike (S) protein inhibitors, that outlines the main fusion site among coronavirus and host cells. Notwithstanding, the Heptad Repeat 1 (HR1) domain on the glycosylated spike (S) protein is the region with less mutability and then the most encouraging target for new inhibitors drugs.The novelty of the proposed approach, compared to others, lies in a precise training of a deep neural network toward the 2019-nCoV virus. Where a Siamese Neural Network (SNN) has been trained to distingue the whole 2019-nCoV protein sequence amongst two different viruses family such as HIV-1 and Ebola. In this way, the present deep learning system has precise knowledge of peptide linkage among 2019-nCoV protein structure and differently, of other works, is not trivially trained on public datasets that have not been provided any ligand-peptide information for 2019-nCoV. Suddenly, the SNN shows a sensitivity of $83\%$ of peptide affinity classification, where $3027$ peptides on SATPdb bank have been tested towards the specific region HR1 of 2019-nCoV exhibiting an affinity of $93\%$ for the peptidyl-prolyl cis-trans isomerase (PPIase) peptide. This affinity between PPIase and HR1 can open new horizons of research since several scientific papers have already shown that CsA immunosuppression drug, a main inhibitor of PPIase, suppress the reproduction of different CoV virus included SARS-CoV and MERS-CoV. Finally, to ensure the scientific reproducibility, code and data have been made public at the following link: https://github.com/bionick87/2019-nCoV

negative: Metagenomic analysis reveals clinical SARS-CoV-2 infection and bacterial or viral superinfection and colonization BACKGROUND: More than two months separated the initial description of SARS-CoV-2 and discovery of its widespread dissemination in the United States. Despite this lengthy interval, implementation of specific quantitative reverse transcription (qRT)-PCR-based SARS-CoV-2 tests in the US has been slow, and testing is still not widely available. Metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence. METHODS: To evaluate metagenomic approaches in the context of the current SARS-CoV-2 epidemic, laboratory-confirmed positive and negative samples from Seattle, Washington were evaluated by metagenomic sequencing, with comparison to a 2019 reference genomic database created before the emergence of SARS-CoV-2. RESULTS: Within 36 hours our results showed clear identification of a novel human Betacoronavirus, closely related to known Betacoronaviruses of bats, in laboratory-proven cases of SARS-CoV-2. A subset of samples also showed superinfection or colonization with human parainfluenza virus 3 or Moraxella species, highlighting the need to test directly for SARS-CoV-2 as opposed to ruling out an infection using a viral respiratory panel. Samples negative for SARS-CoV-2 by RT-PCR were also negative by metagenomic analysis, and positive for Rhinovirus A and C. Unlike targeted SARS-CoV-2 qRT-PCR testing, metagenomic analysis of these SARS-CoV-2 negative samples identified candidate etiological agents for the patients’ respiratory symptoms. CONCLUSION: Taken together, these results demonstrate the value of metagenomic analysis in the monitoring and response to this and future viral pandemics.

negative: Insights into the Recent 2019 Novel Coronavirus (SARS-CoV-2) in Light of Past Human Coronavirus Outbreaks Coronaviruses (CoVs) are RNA viruses that have become a major public health concern since the Severe Acute Respiratory Syndrome-CoV (SARS-CoV) outbreak in 2002. The continuous evolution of coronaviruses was further highlighted with the emergence of the Middle East Respiratory Syndrome-CoV (MERS-CoV) outbreak in 2012. Currently, the world is concerned about the 2019 novel CoV (SARS-CoV-2) that was initially identified in the city of Wuhan, China in December 2019. Patients presented with severe viral pneumonia and respiratory illness. The number of cases has been mounting since then. As of late February 2020, tens of thousands of cases and several thousand deaths have been reported in China alone, in addition to thousands of cases in other countries. Although the fatality rate of SARS-CoV-2 is currently lower than SARS-CoV, the virus seems to be highly contagious based on the number of infected cases to date. In this review, we discuss structure, genome organization, entry of CoVs into target cells, and provide insights into past and present outbreaks. The future of human CoV outbreaks will not only depend on how the viruses will evolve, but will also depend on how we develop efficient prevention and treatment strategies to deal with this continuous threat.

negative: Molecular Mechanism of Evolution and Human Infection with SARS-CoV-2 The outbreak of a novel coronavirus, which was later formally named the severe acute respiratory coronavirus 2 (SARS-CoV-2), has caused a worldwide public health crisis. Previous studies showed that SARS-CoV-2 is highly homologous to SARS-CoV and infects humans through the binding of the spike protein to ACE2. Here, we have systematically studied the molecular mechanisms of human infection with SARS-CoV-2 and SARS-CoV by protein-protein docking and MD simulations. It was found that SARS-CoV-2 binds ACE2 with a higher affinity than SARS-CoV, which may partly explain that SARS-CoV-2 is much more infectious than SARS-CoV. In addition, the spike protein of SARS-CoV-2 has a significantly lower free energy than that of SARS-CoV, suggesting that SARS-CoV-2 is more stable and may survive a higher temperature than SARS-CoV. This provides insights into the evolution of SARS-CoV-2 because SARS-like coronaviruses have originated in bats. Our computation also suggested that the RBD-ACE2 binding for SARS-CoV-2 is much more temperature-sensitive than that for SARS-CoV. Thus, it is expected that SARS-CoV-2 would decrease its infection ability much faster than SARS-CoV when the temperature rises. These findings would be beneficial for the disease prevention and drug/vaccine development of SARS-CoV-2.

negative: A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (< 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 90.55% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 {+/-} 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 {+/-} 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geo-localization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.

negative: Putative hAPN receptor binding sites in SARS_CoV spike protein. AIM To obtain the information of ligand-receptor binding between the S protein of SARS-CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites in the S protein of SARS-CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS-CoV in validating the bioinformatics predictions. RESULTS Possible binding sites in the SARS-CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS-CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION CD13 may be a possible receptor of the SARS-CoV S protein, which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

negative: Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions The COVID-19 pandemic has spread across the globe at an alarming rate in the last four months. However, unlike any of the previous global outbreaks the availability of hundreds of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 480 full-length (>29000 nt) sequences from the 1575 SARS-CoV-2 sequences available and identified 37 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, two of the 37 substitutions occur within highly conserved secondary structures in the 5’ and 3’ untranslated regions that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define 3 mutually exclusive lineages (A1, B1 and C1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2.

negative: Severe acute respiratory syndrome-associated coronavirus genotype and its characterization. OBJECTIVE To study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics. METHODS A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done. RESULTS By bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively. CONCLUSION Attention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.

negative: Development and clinical application of a rapid and sensitive loop-mediated isothermalamplification test for SARS-CoV-2 infection Background The outbreak of SARS-CoV-2 urgently requires sensitive and convenient COVID-19 diagnostics to assure the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2 in both qualified laboratories and point-of-care settings. Methods Patients with suspected COVID-19 and close contacts between Jan 26 and April 8, 2020, were recruited from two hospitals. Respiratory samples were collected and tested with LAMP and the results were compared with those obtained by RT-qPCR. The inconsistent samples between these two methods were subjected to next-generation sequencing for confirmation. In addition, we tested the RT-LAMP on an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Results We finally collected a cohort of 129 cases (329 nasopharyngeal swabs) and the independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). RT-LAMP was validated to be accurate (overall sensitivity and specificity: 88.89% and 99.00%; positive and negative predictive values: 94.74% and 97.78%) and diagnostically useful (positive and negative likelihood ratios: 88.89 and 0.11). RT-LAMP showed an increased sensitivity (88.89% vs 81.48%) and high consistency (kappa 0.92) compared with RT-qPCR for SARS-CoV-2 screening while requiring only constant temperature heating and visual inspection. The median time required for RT-LAMP was less than 1 h from sample to result. Further analyses indicated that RT-LAMP was feasible for asymptomatic patients and did not cross-react with other respiratory pathogen infections. Conclusion The RT-LAMP assay offers a rapid, sensitive and straightforward detection for SARS-CoV-2 infection, which could aid the expansion of COVID-19 testing in the public domain and hospitals.

negative: Epidemiological and phylogenetic analysis of canine kobuviruses in Tangshan, China Canine kobuviruses (CaKoV) have been found in healthy and diarrheic dogs as well as asymptomatic wild carnivores in various countries. In order to investigate the prevalence and evolution of CaKoV in Tangshan, China, 82 dog fecal samples from pet hospitals in Tangshan were subjected to RT-PCR targeting a segment of the 3D gene of CaKoV. Using this method, we identified CaKoV in 14 samples (17.07%, 14/82). Of the CaKoV-positive samples, 78.57% (11/14) and 50% (7/14) were positive for canine parvovirus and canine coronavirus, respectively. The nucleotide sequences of the 14 strains 96.6%–100% identical to each other and 77.6%–99.2% identical to representative sequences from the NCBI GenBank database. We also amplified the 14 VP1 gene sequences and found that they were 93.3%–99.6% identical to each other and 73.3%–97.8% identical to representative sequences from the NCBI GenBank database. Phylogenetic analysis revealed that the 14 CaKoV strains from Tangshan are closely related to those identified in China and Thailand and display less similarity to those found in Africa, the United States, and Europe. Our data suggest that CaKoV circulated in young pet dogs in Tangshan and displays a high co-infection rate with CCoV and CPV. However, the relationship between the three viruses and their roles in the host requires further investigation.

negative: Complete Genome Sequences of Five Human Coronavirus NL63 Strains Causing Respiratory Illness in Hospitalized Children in China We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.

negative: An 81 base-pair deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona (Jan-Mar 2020) On January 26 2020, the first Coronavirus Disease 2019 (COVID-19) case was reported in Arizona of an individual with travel history (3rd case in the US). Here, we report on early SARS-CoV-2 sentinel surveillance in Tempe, Arizona. Genomic characterization identified an isolate encoding a 27 amino acid in-frame deletion in accessory protein ORF7a, the ortholog of SARS-CoV immune antagonist ORF7a/X4 .

negative: Genomic characterization of a novel SARS-CoV-2 A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with human to human transmission and extreme human sickness has been as of late announced from the city of Wuhan in China. Our objectives were to mutation analysis between recently reported genomes at various times and locations and to characterize the genomic structure of SARS-CoV-2 using bioinformatics programs. Information on the variation of viruses is of considerable medical and biological impacts on the prevention, diagnosis, and therapy of infectious diseases. To understand the genomic structure and variations of the SARS-CoV-2. The study analyzed 95 SARS-CoV-2 complete genome sequences available in GenBank, National MicrobiologyData Center (NMDC) and NGDC Genome Warehouse from December-2019 until 05 of April-2020. The genomic signature analysis demonstrates that a strong association between the time of sample collection, location of sample and accumulation of genetic diversity. We found 116 mutations, the three most common mutations were 8782C>T in ORF1ab gene, 28144T>C in ORF8 gene and 29095C>T in the N gene. The mutations might affect the severity and spread of the SARS-CoV-2. The finding heavily supports an intense requirement for additional prompt, inclusive investigations that combine genomic detail, epidemiological information and graph records of the clinical features of patients with COVID-19.

negative: One-year molecular survey of astrovirus infection in turkeys in Poland The presence of turkey astrovirus (TAstV) was monitored in meat-type turkey flocks in Poland in 2008. Clinical samples (10 individual faecal swabs/flock) from 77 flocks aged 1-19 weeks were collected from different regions of the country. RT-PCR experiments were performed for detection and molecular characterization of TAstV using four sets of primers within the RdRp gene (ORF1b). The prevalence of astrovirus was 34/77 (44.15%) in the flocks tested. TAstV type 2 was associated with 30 of 77 infections (38.9%), either alone or in mixed infections; TAstV type 1 was detected in 9 of 77 flocks (11.6%), either alone or in mixed infections; ANV was detected only in one flock (1.29%) by sequence analysis during this study. Phylogenetic analysis revealed genetic variability in the TAstV strains that were isolated. Some of Polish TAstV-2 strains were genetically related to the North American isolates; however, most of them formed a distinct subgroup of “European” isolates, suggesting their separate origin or evolution. Additionally, due to the high variability of the TAstV sequences, the most suitable method for TAstV typing seems to be sequencing.

negative: Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 5 2020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

negative: Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies The recent severe acute respiratory syndrome, known as Corona Virus Disease 2019 (COVID-19) has spread so much rapidly and severely to induce World Health Organization (WHO) to declare state of emergency over the new coronavirus SARS-CoV-2 pandemic. While several countries have chosen the almost complete lock-down for slowing down SARS-CoV-2 spread, scientific community is called to respond to the devastating outbreak by identifying new tools for diagnosis and treatment of the dangerous COVID-19. With this aim we performed an in silico comparative modeling analysis, which allows to gain new insights about the main conformational changes occurring in the SARS-CoV-2 spike protein, at the level of the receptor binding domain (RBD), along interactions with human cells angiotensin converting enzyme 2 (ACE2) receptor, that favour human cell invasion. Furthermore, our analysis provides i) an ideal pipeline to identify already characterized antibodies that might target SARS-CoV-2 spike RBD, for preventing interactions with the human ACE2, and ii) instructions for building new possible neutralizing antibodies, according to chemical/physical space restraints and complementary determining regions (CDR) mutagenesis of the identified existing antibodies. The proposed antibodies show in silico a high affinity for SARS-CoV-2 spike RBD and can be used as reference antibodies also for building new high affinity antibodies against present and future coronavirus able to invade human cells through interactions of their spike proteins with the human ACE2. More in general, our analysis provides indications for the set-up of the right biological molecular context for investigating spike RBD-ACE2 interactions for the development of new vaccines, diagnosis kits and other treatments based on the usage or the targeting of SARS-CoV-2 spike protein.

negative: Coronavirus Pandemic—Therapy and Vaccines The current coronavirus COVID-19 pandemic, which originated in Wuhan, China, has raised significant social, psychological and economic concerns in addition to direct medical issues. The rapid spread of severe acute respiratory syndrome-coronavirus (SARS-CoV)-2 to almost every country on the globe and the failure to contain the infections have contributed to fear and panic worldwide. The lack of available and efficient antiviral drugs or vaccines has further worsened the situation. For these reasons, it cannot be overstated that an accelerated effort for the development of novel drugs and vaccines is needed. In this context, novel approaches in both gene therapy and vaccine development are essential. Previous experience from SARS- and MERS-coronavirus vaccine and drug development projects have targeted glycoprotein epitopes, monoclonal antibodies, angiotensin receptor blockers and gene silencing technologies, which may be useful for COVID-19 too. Moreover, existing antivirals used for other types of viral infections have been considered as urgent action is necessary. This review aims at providing a background of coronavirus genetics and biology, examples of therapeutic and vaccine strategies taken and potential innovative novel approaches in progress.

negative: Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor The 2002–3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history(1). An ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV)(2) suggests that this group of viruses remains a major threat and that their distribution is wider than previously recognized. Although bats have been suggested as the natural reservoirs of both viruses(3–5), attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa(5–8), but none are considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins (S) to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2)(9,10). Here, we report whole genome sequences of two novel bat CoVs from Chinese horseshoe bats (Family: Rhinolophidae) in Yunnan, China; RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat CoVs, particularly in the receptor binding domain (RDB) of the S protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat fecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses the ACE2s from human, civet and Chinese horseshoe bat for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

negative: Severe acute respiratory syndrome (SARS)—paradigm of an emerging viral infection Abstract An acute and often severe respiratory illness emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS) came to an end in July 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. Starting in March 2003, the World Health Organization (WHO) organised an unprecedented international effort by leading laboratories working together to find the causative agent. Little more than one week later, three research groups from this WHO-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in SARS patients, using different approaches. After Koch’s postulates had been fulfilled, WHO officially declared on 16 April 2003 that this virus never before seen in humans is the cause of SARS. Ever since, progress around SARS-associated coronavirus (SARS-CoV) has been swift. Within weeks of the first isolate being obtained, its complete genome was sequenced. Diagnostic tests based on the detection of SARS-CoV RNA were developed and made available freely and widely; nevertheless the SARS case definition still remains based on clinical and epidemiological criteria. The agent’s environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. Despite its grave consequences in humanitarian, political and economic terms, SARS may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more “traditional” techniques carried out to an excellent standard.

negative: Characterization and phylogenetic analysis of new bat astroviruses detected in Gabon, Central Africa. Astroviruses are emerging RNA viruses that cause enteropathogenic infections in humans and in other mammals. The identification of astroviruses in a wide range of animals highlights the zoonotic importance of these viruses. Bats can harbor many different viruses, among which some are highly pathogenic for humans (for instance, Nipah, Ebola and SARS coronavirus), and also several astroviruses. As some RNA viruses can be directly transmitted from bats to humans, it is crucial to collect data about their frequency, genetic diversity and phylogenetic characterization. In this study, we report the molecular identification of 44 new astroviruses (with a detection rate of 4.5%) in 962 apparently healthy bats that belong to five different species and that were captured in different caves in North-East Gabon, Central Africa. Our results show that bat astroviruses form a group that is genetically distinct from astroviruses infecting other mammals. Moreover, these astroviruses showed an important genetic diversity and low host restriction in bat species.

negative: The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39). The complete genomic nucleotide sequence (29.7kb) of a Hong Kong severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) strain HK-39 is determined. Phylogenetic analysis of the genomic sequence reveals it to be a distinct member of the Coronaviridae family. 5' RACE assay confirms the presence of at least six subgenomic transcripts all containing the predicted intergenic sequences. Five open reading frames (ORFs), namely ORF1a, 1b, S, M, and N, are found to be homologues to other CoV members, and three more unknown ORFs (X1, X2, and X3) are unparalleled in all other known CoV species. Optimal alignment and computer analysis of the homologous ORFs has predicted the characteristic structural and functional domains on the putative genes. The overall nucleotides conservation of the homologous ORFs is low (<5%) compared with other known CoVs, implying that HK-39 is a newly emergent SARS-CoV phylogenetically distant from other known members. SimPlot analysis supports this finding, and also suggests that this novel virus is not a product of a recent recombinant from any of the known characterized CoVs. Together, these results confirm that HK-39 is a novel and distinct member of the Coronaviridae family, with unknown origin. The completion of the genomic sequence of the virus will assist in tracing its origin.

negative: The R Protein of SARS-CoV: Analyses of Structure and Function Based on Four Complete Genome Sequences of Isolates BJ01-BJ04 The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.

negative: Interhuman transmissibility of Middle East respiratory syndrome coronavirus: estimation of pandemic risk BACKGROUND: The new Middle East respiratory syndrome coronavirus (MERS-CoV) infection shares many clinical, epidemiological, and virological similarities with that of severe acute respiratory syndrome (SARS)-CoV. We aimed to estimate virus transmissibility and the epidemic potential of MERS-CoV, and to compare the results with similar findings obtained for prepandemic SARS. METHODS: We retrieved data for MERS-CoV clusters from the WHO summary and subsequent reports, and published descriptions of cases, and took into account 55 of the 64 laboratory-confirmed cases of MERS-CoV reported as of June 21, 2013, excluding cases notified in the previous 2 weeks. To assess the interhuman transmissibility of MERS-CoV, we used Bayesian analysis to estimate the basic reproduction number (R(0)) and compared it to that of prepandemic SARS. We considered two scenarios, depending on the interpretation of the MERS-CoV cluster-size data. RESULTS: With our most pessimistic scenario (scenario 2), we estimated MERS-CoV R(0) to be 0·69 (95% CI 0·50–0·92); by contrast, the R(0) for prepandemic SARS-CoV was 0·80 (0·54–1·13). Our optimistic scenario (scenario 1) yielded a MERS-CoV R(0) of 0·60 (0·42–0·80). Because of recent implementation of effective contact tracing and isolation procedures, further MERS-CoV transmission data might no longer describe an entire cluster, but only secondary infections directly caused by the index patient. Hence, we calculated that, under scenario 2, eight or more secondary infections caused by the next index patient would translate into a 5% or higher chance that the revised MERS-CoV R(0) would exceed 1—ie, that MERS-CoV might have pandemic potential. INTERPRETATION: Our analysis suggests that MERS-CoV does not yet have pandemic potential. We recommend enhanced surveillance, active contact tracing, and vigorous searches for the MERS-CoV animal hosts and transmission routes to human beings. FUNDING: Agence Nationale de la Recherche (Labex Integrative Biology of Emerging Infectious Diseases), and the European Community's Seventh Framework Programme project PREDEMICS.

negative: Machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: COVID-19 case study The 2019 novel coronavirus (renamed SARS-CoV-2, and generally referred to as the COVID-19 virus) has spread to 184 countries with over 1.5 million confirmed cases. Such major viral outbreaks demand early elucidation of taxonomic classification and origin of the virus genomic sequence, for strategic planning, containment, and treatment. This paper identifies an intrinsic COVID-19 virus genomic signature and uses it together with a machine learning-based alignment-free approach for an ultra-fast, scalable, and highly accurate classification of whole COVID-19 virus genomes. The proposed method combines supervised machine learning with digital signal processing (MLDSP) for genome analyses, augmented by a decision tree approach to the machine learning component, and a Spearman’s rank correlation coefficient analysis for result validation. These tools are used to analyze a large dataset of over 5000 unique viral genomic sequences, totalling 61.8 million bp, including the 29 COVID-19 virus sequences available on January 27, 2020. Our results support a hypothesis of a bat origin and classify the COVID-19 virus as Sarbecovirus, within Betacoronavirus. Our method achieves 100% accurate classification of the COVID-19 virus sequences, and discovers the most relevant relationships among over 5000 viral genomes within a few minutes, ab initio, using raw DNA sequence data alone, and without any specialized biological knowledge, training, gene or genome annotations. This suggests that, for novel viral and pathogen genome sequences, this alignment-free whole-genome machine-learning approach can provide a reliable real-time option for taxonomic classification.

negative: Access to Care for Dementia patients suffering from COVID-19

negative: When Darkness Becomes a Ray of Light in the Dark Times: Understanding the COVID-19 via the Comparative Analysis of the Dark Proteomes of SARS-CoV-2, Human SARS and Bat SARS-Like Coronaviruses Recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 9,00,000 cases of SARS-CoV-2 infection and more than 47,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted that the COVID-19 is a pandemic now. The multiple sequence alignment data correlated with the already published reports on the SARS-CoV-2 evolution and indicated that this virus is closely related to the bat Severe Acute Respiratory Syndrome-like coronavirus (bat SARS-like CoV) and the well-studied Human SARS coronavirus (SARS CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for Nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins were shown to contain molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of the SARS-CoV-2 proteome will have important implications for the structural and non-structural biology of SARS or SARS-like coronaviruses. Significance The infection caused by a novel coronavirus (SARS-CoV-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current COVID-19 pandemic. No in-depth information on structures and functions of SARS-CoV-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. Our study provides the first comparative analysis of the order- and disorder-based features of the SARS-CoV-2 proteome relative to human SARS and bat CoV that may be useful for structure-based drug discovery.

negative: Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

negative: A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation. Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

negative: Structural basis of receptor recognition by SARS-CoV-2 A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.

negative: Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections.

negative: Etiology and genetic evolution of canine coronavirus circulating in five provinces of China, during 2018–2019 Abstract As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV–I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

negative: Non-invasive surveys of mammalian viruses using environmental DNA Environmental DNA (eDNA) and its subdiscipline, invertebrate-derived DNA (iDNA) have been used to survey biodiversity non-invasively [1,2]. Water is ubiquitous in most ecosystems, and, among invertebrates, terrestrial haematophagous leeches are abundant and can be easily collected in many tropical rainforests [3,4]. Such non-invasive nucleic acid sources can mitigate difficulties of obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA sources have been applied to monitoring specific wildlife pathogens [5,6]. However, previous studies have focused on known pathogens, whereas most wildlife pathogens are uncharacterized and unknown. Non-invasive approaches to monitoring known and novel pathogens may be of particular benefit in ecosystems prone to viral emergence, many of which occur in areas where invasive sampling is challenging, such as tropical rainforests. Here, we show that both eDNA from natural waterholes, and iDNA from terrestrial haematophagous leeches, can be used to detect unknown viruses circulating in mammalian hosts (Figure 1). Using a curated set of RNA oligonucleotides based on the ViroChip microarray assay [7] as baits in a hybridization capture system, multiple mammalian RNA and DNA viruses were detected from both eDNA and iDNA samples. Congruence was found between host DNA assignment and viruses identified in leeches, and between animals observed visiting the waterholes and the viruses detected. Our results demonstrate that eDNA/iDNA samples may represent an effective non-invasive resource for studying wildlife viral diversity. Several of the detected viruses were novel, highlighting the potential of eDNA/iDNA for epidemiological analysis of emerging viruses prior to their emergence. Highlights Environmental DNA (water and blood-sucking leeches) provided a non-invasive method of screening wildlife for viruses A comprehensive viral RNA oligonucleotide bait set was developed to capture known and unknown mammalian virus diversity Leech blood meal host determination and viruses identified were congruent Viruses determined from water correlated with known and observed species visiting the water sources In brief Alfano, Dayaram, et al. demonstrate that environmental DNA from southeast Asian leech bloodmeals and waterholes from Africa and Mongolia can be used as to detect viruses circulating in wildlife. These nucleic acid sources may represent an effective non-invasive resource for studying wildlife viral diversity and emerging viruses pre-emergence.

negative: Genomic Sequencing of the Severe Acute Respiratory Syndrome-Coronavirus The polymerase chain reaction (PCR), which can exponentially replicate a target DNA sequence, has formed the basis for the sensitive and direct examination of clinical samples for evidence of infection. During the epidemic of severe acute respiratory syndrome (SARS) in 2003, PCR not only offered a rapid way to diagnose SARS-coronavirus (SARS-CoV) infection, but also made the molecular analysis of its genomic sequence possible. Sequence variations were observed in the SAR-CoV obtained from different patients in this epidemic. These unique viral genetic signatures can be applied as a powerful molecular tool in tracing the route of transmission and in studying the genome evolution of SARS-CoV. To extract this wealth of information from the limited primary clinical specimens of SARS patients, we were presented with the challenge of efficiently amplifying fragments of the SARS-CoV genome for analysis. In this chapter, we will discuss how we managed to accomplish this task with our optimized protocols on reversetranscription, nested PCR amplification, and DNA cycle sequencing. We will also discuss the sequence variations that typified some strains of SARS-CoV in the different phases during this epidemic. PCR amplification of the viral sequence and genomic sequencing of these critical sequence variations of re-emerging SARS-CoV strains would give us quick insights into the virus.

negative: SARS-CoV-2 and human milk: what is the evidence? The novel coronavirus SARS-CoV-2 has emerged as one of the most compelling public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical, and public health communities have rapidly engaged to find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS-CoV-2 transmission. While respiratory droplets are a known mechanism of transmission, other mechanisms are possible. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronavirus (including SARS-CoV-2) via human milk and/or breastfeeding. There is a single study providing some evidence of vertical transmission of human coronavirus 229E, a single study evaluating presence of SARS-CoV in human milk (it was negative), and no published data on MERS-CoV and human milk. There are 9 case studies of human milk tested for SARS-CoV-2; none detected the virus. Importantly, none of the published studies on coronaviruses and human milk report validation of their analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, SARS-CoV-2) during breastfeeding are discussed.

negative: CoV-Seq: SARS-CoV-2 Genome Analysis and Visualization Summary COVID-19 has become a global pandemic not long after its inception in late 2019. SARS-CoV-2 genomes are being sequenced and shared on public repositories at a fast pace. To keep up with these updates, scientists need to frequently refresh and reclean datasets, which is ad hoc and labor-intensive. Further, scientists with limited bioinformatics or programming knowledge may find it difficult to analyze SARS-CoV-2 genomes. In order to address these challenges, we developed CoV-Seq, a webserver to enable simple and rapid analysis of SARS-CoV-2 genomes. Given a new sequence, CoV-Seq automatically predicts gene boundaries and identifies genetic variants, which are presented in an interactive genome visualizer and are downloadable for further analysis. A command-line interface is also available for high-throughput processing. Availability and Implementation CoV-Seq is implemented in Python and Javascript. The webserver is available at http://covseq.baidu.com/ and the source code is available from https://github.com/boxiangliu/covseq. Contact jollier.liu@gmail.com Supplementary information Supplementary information are available at bioRxiv online.

negative: Serology of Severe Acute Respiratory Syndrome: Implications for Surveillance and Outcome Background. Severe acute respiratory syndrome (SARS) is a novel infectious disease. No information is currently available on host-specific immunity against the SARS coronavirus (CoV), and detailed characteristics of the epidemiology of SARS CoV infection have not been identified. Methods. ELISA was used to detect antibody to SARS CoV. Reverse-transcriptase polymerase chain reaction was used to detect SARS CoV RNA. T cells in peripheral blood of patients were quantified by flow cytometry. Results. Of 36 patients with probable SARS CoV infection, 30 (83.3%) were positive for IgG antibody to SARS CoV; in contrast, only 3 of 48 patients with suspected SARS CoV infection, 0 of 112 patients with fever but without SARS, and 0 of 96 healthy control individuals were positive for it. IgG antibody to SARS CoV was first detected between day 5 and day 47 after onset of illness (mean ± SD, 18.7±10.4). Conclusion. Detection of antibody to SARS CoV is useful in the diagnosis of SARS; however, at the incubation and initial phases of the illness, serological assay is of little value, because of late seroconversion in most patients.

negative: Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

negative: Tracking the genomic footprints of SARS-CoV-2 transmission Abstract There is considerable public and scientific interest on the origin, spread and evolution of SARS-CoV-2. A recent study by Lu et al. [1], conducted genomic sequencing and analysis of SARS-CoV-2 in Guangdong, revealing its early transmission out of Hubei and shedding light on the effectiveness of controlling local transmission chains.

negative: Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction Abstract The severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein is an abundant virion protein, and its interaction with the nucleocapsid protein is crucial for viral assembly and morphogenesis. Although the interacting region in the nucleocapsid protein was mapped to residues 168–208, the interacting region in the membrane protein and the interaction nature are still unclear. In this work, by using yeast two-hybrid and surface plasmon resonance techniques, the residues 197–221 of the membrane protein and the residues 351–422 of the nucleocapsid protein were determined to be involved in their interaction. Sequence analysis revealed that these two fragments are highly charged at neutral pH, suggesting that their interaction may be of ionic nature. Kinetic assays indicated that the endodomain (aa102–221) of the membrane protein interacts with the nucleocapsid protein with high affinity (K D =0.55±0.04μM), however, this interaction could be weakened greatly by acidification, higher salt concentration (400mM NaCl) and divalent cation (50mM Ca2+), which suggests that electrostatic attraction might play an important role in this interaction. In addition, it is noted that two highly conserved amino acids (L218 and L219) in the membrane protein are not involved in this interaction. Here, we show that electrostatic interactions between the carboxyl termini of SARS-CoV membrane protein and nucleocapsid protein largely mediate the interaction of these two proteins. These results might facilitate therapeutic strategies aiming at the disruption of the association between SARS-CoV membrane and nucleocapsid proteins.

negative: A virus that has gone viral: amino acid mutation in S protein of Indian isolate of Coronavirus COVID-19 might impact receptor binding, and thus, infectivity Since 2002, β coronaviruses (CoVs) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the recent outbreak of SARS-CoV-2 late in 2019 (also named as COVID-19 or novel coronavirus 2019 or nCoV2019). Spike (S) protein, one of the structural proteins of this virus plays key role in receptor (ACE2) binding and thus virus entry. Thus, this protein has attracted scientists for detailed study and therapeutic targeting. As the nCoV2019 takes its course throughout the world, more and more sequence analyses are being done and genome sequences are being deposited in various databases. From India, two clinical isolates have been sequenced and the full genome has been deposited in GenBank. We have performed sequence analyses of the Spike protein of the Indian isolates and compared with that of the Wuhan, China (where the outbreak was first reported). While all the sequences of Wuhan isolates are identical, we found point mutations in the Indian isolates. Out of the two isolates, one was found to harbor a mutation in its receptor-binding domain (RBD) at position 407. At this site, arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic amino acid that is also a C-β branched amino acid). This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor binding of the virus. Although this finding needs further validation and more sequencing, the information might be useful in rational drug designing and vaccine engineering.

negative: Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA BACKGROUND: Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates. METHODOLOGY/PRINCIPAL FINDINGS: Sequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice. CONCLUSIONS/SIGNIFICANCE: These data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus.

negative: Laboratory Diagnosis of Novel Coronavirus Disease 2019 (COVID-19) Infection Coronavirus disease 2019 (COVID-19) is an infection caused by the novel coronavirus severe acute respiratory coronavirus 2 (SARS-CoV-2). The infection manifests as a mild flu to severe acute respiratory infection. The World Health Organization (WHO) declared COVID-19 as a global pandemic on March 11, 2020. The disease spreads by droplet infection from person to person. Early diagnosis is the key for prompt management of cases and control of the spread of the virus. Currently, the laboratory diagnosis of SARS-CoV-2 is based on nucleic acid amplification tests (NAAT) like real-time reverse transcriptase (RT-PCR). Various genes like E, N, S, ORF and RdRp are targeted as a part of screening and confirmation of cases. Furthermore, nucleic acid sequencing may be done for the identification of mutation in the genome of SARS-CoV-2. The development of serological assays and point of care molecular test will further intensify the diagnostic modalities of SARS-CoV-2.

negative: Identification of SARS-CoV RBD-targeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2. SARS-CoV-2-caused COVID-19 cases are growing globally, calling for developing effective therapeutics to control the current pandemic. SARS-CoV-2 and SARS-CoV recognize angiotensin-converting enzyme 2 (ACE2) receptor via the receptor-binding domain (RBD). Here, we identified six SARS-CoV RBD-specific neutralizing monoclonal antibodies (nAbs) that cross-reacted with SARS-CoV-2 RBD, two of which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing epitopes on RBD did not overlap with the ACE2-binding sites, whereas those recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. Our study provides an alternative approach to prevent SARS-CoV-2 infection using anti-SARS-CoV nAbs.

negative: Endoscopic anatomy of the cervical vertebral canal in the horse: a cadaver study. REASON FOR PERFORMING STUDY Localisation of spinal cord compression in horses with cervical vertebral stenotic myelopathy is inexact. Vertebral canal endoscopy has been used in man to localise spinal cord lesions and has the potential to become a useful diagnostic technique in horses. OBJECTIVE To establish a surgical approach via the atlanto-occipital space to the cervical vertebral canal in equine cadavers and describe the endoscopic anatomy of the cervical epidural and subarachnoid spaces. METHODS The cadavers of 25 mature horses were used to assess 3 surgical methods to approach the cervical vertebral canal, including 2 minimally invasive and one open technique. Once the approach had been made, a flexible videoendoscope was inserted into the epidural space (epiduroscopy) or the subarachnoid space (myeloscopy) and advanced caudally until the intervertebral space between C7 and T1 was reached. RESULTS The epidural and subarachnoid spaces could not be accessed reliably using the minimally invasive techniques. Furthermore, damage to the nervous tissues was a frequent complication with these procedures. The open approach allowed successful insertion of the videoendoscope into the epidural and subarachnoid spaces in all horses and no inadvertent damage was observed. Anatomical structures that could be seen in the epidural space included the dura mater, nerve roots, fat and the ventral internal vertebral venous plexus. In the subarachnoid space, the spinal cord, nerve roots, blood vessels, denticulate ligaments and external branch of the accessory nerve were seen. CONCLUSIONS Using the open approach, epiduroscopy and myeloscopy over the entire length of the cervical vertebral canal are possible in the mature horse. POTENTIAL RELEVANCE Cervical vertebral canal endoscopy may become a valuable tool to localise the site of spinal cord injury in horses with cervical vertebral stenotic myelopathy and could aid in the diagnosis of other diseases of the cervical spinal cord.

negative: Phylogenetic and recombination analysis of Tobacco bushy top virus in China BACKGROUND: During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates. METHODS: 5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. RESULTS: The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations. CONCLUSIONS: The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0340-2) contains supplementary material, which is available to authorized users.

negative: An updated analysis of variations in SARS-CoV-2 genome A novel pathogen, named SARS-CoV-2, has caused an unprecedented worldwide pandemic in the first half of 2020. As the SARS-CoV-2 genome sequences have become available, one of the important focus of scientists has become tracking variations in the viral genome. In this study, 30366 SARS-CoV-2 isolate genomes were aligned using the software developed by our group (ODOTool) and 11 variations in SARS-CoV-2 genome over 10% of whole isolates were discussed. Results indicated that, frequency rates of these 11 variations change between 3.56%–88.44 % and these rates differ greatly depending on the continents they have been reported. Despite some variations being in low frequency rate in some continents, C14408T and A23403G variations on Nsp12 and S protein, respectively, observed to be the most prominent variations all over the world, in general, and both cause missense mutations. It is also notable that most of isolates carry C14408T and A23403 variations simultaneously and also nearly all isolates carrying the G25563T variation on ORF3a, also carry C14408T and A23403 variations, although their location distributions are not similar. All these data should be considered towards development of vaccine and antiviral treatment strategies as well as tracing diversity of virus in all over the world.

negative: A porcine enterovirus G associated with enteric disease contains a novel papain-like cysteine protease Identification of unknown pathogens in pigs displaying enteric illness is difficult due to the large diversity of bacterial and viral species found within faecal samples. Current methods often require bacterial or viral isolation, or testing only a limited number of known species using quantitative PCR analysis. Herein, faeces from two 25-day-old piglets with diarrhoea from Texas, USA, were analysed by metagenomic next-generation sequencing to rapidly identify possible pathogens. Our analysis included a bioinformatics pipeline of rapid short-read classification and de novo genome assembly which resulted in the identification of a porcine enterovirus G (EV-G), a complete genome with substantial nucleotide differences (>30 %) among current sequences, and a novel non-structural protein similar in sequence to the Torovirus papain-like cysteine protease (PL(pro)). This discovery led to the identification and circulation of an EV-G with a novel PL(pro) in the USA that has not been previously reported.

negative: European Hedgehogs as Hosts for Borrelia spp., Germany

negative: Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented during March 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.

negative: Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 site Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct 4 amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability and transmission.

negative: Molecular architecture of the SARS-CoV-2 virus Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins and the complexes of the spike (S) proteins with the cellular receptor ACE2 or neutralizing antibodies, detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and postfusion conformations were determined to average resolutions of 9-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The in situ architecture of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. These characterizations have revealed the architecture of the SARS-CoV-2 virus to an unprecedented resolution, and shed lights on how the virus packs its ~30 Kb long single-segmented RNA in the ~80 nm diameter lumen. Overall, the results unveiled the molecular architecture and assembly of the SARS-CoV-2 in native context.

negative: Rapid implementation of real-time SARS-CoV-2 sequencing to investigate healthcare-associated COVID-19 infections Background The burden and impact of healthcare-associated COVID-19 infections is unknown. We aimed to examine the utility of rapid sequencing of SARS-CoV-2 combined with detailed epidemiological analysis to investigate healthcare-associated COVID-19 infections and to inform infection control measures. Methods We set up rapid viral sequencing of SARS-CoV-2 from PCR-positive diagnostic samples using nanopore sequencing, enabling sample-to-sequence in less than 24 hours. We established a rapid review and reporting system with integration of genomic and epidemiological data to investigate suspected cases of healthcare-associated COVID-19. Results Between 13 March and 24 April 2020 we collected clinical data and samples from 5191 COVID-19 patients in the East of England. We sequenced 1000 samples, producing 747 complete viral genomes. We conducted combined epidemiological and genomic analysis of 299 patients at our hospital and identified 26 genomic clusters involving 114 patients. 66 cases (57.9%) had a strong epidemiological link and 15 cases (13.2%) had a plausible epidemiological link. These results were fed back clinical, infection control and hospital management teams, resulting in infection control interventions and informing patient safety reporting. Conclusions We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and demonstrated the benefit of combined genomic and epidemiological analysis for the investigation of healthcare-associated COVID-19 infections. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection control interventions to reduce further healthcare-associated infections.

negative: Full-genome sequences of GII.13[P21] recombinant norovirus strains from an outbreak in Changsha, China On 31 March 2019, 68 school students suffered from vomiting, diarrhea, and abdominal pain after participating in a group activity at a commercial park. In this outbreak, multiple norovirus genotypes were observed, including GII.2[P16], GII.17[P17], and GII.13[P21]. Further, we determined the full-genome sequences of two strains of GII.13[P21] recombinant noroviruses, which were 7434 nt long. Phylogenetic analysis based on open reading frames (ORFs) 1 and 2 revealed that these recombinants were related to stains of different genotypes from different countries. The full genome nucleotide sequences of the two isolates were 97.0% and 98.0% identical to those of strains from London and Thailand, respectively. Simplot analysis revealed the presence of a break point at nt 5059 in the ORF1 region. The histo-blood group antigen binding sites were conserved in both recombinant viruses. Our findings not only provide valuable genetic information about a recombinant norovirus but also contribute to our general understanding of the evolution, genetic diversity, and distribution of noroviruses.

negative: Genomic Sequencing and Analysis of Eight Camel-Derived Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Isolates in Saudi Arabia. Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.

negative: Coronavirus in Continuous Flux: From SARS‐CoV to SARS‐CoV‐2 The world is currently experiencing a global pandemic caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which causes severe respiratory disease similar to SARS. Previous studies have suggested that SARS‐CoV‐2 shares 79% and 96% sequence identity to SARS‐CoV and to bat coronavirus RaTG13, respectively at the whole‐genome level. Furthermore, a series of studies have shown that SARS‐CoV‐2 induces clusters of severe respiratory illnesses (i.e., pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS)) resembling SARS‐CoV. Moreover, the pathological syndrome may, in part, be caused by cytokine storms and dysregulated immune responses. Thus, in this work the recent literature surrounding the biology, clinical manifestations, and immunology of SARS‐CoV‐2 is summarized, with the aim of aiding prevention, diagnosis, and treatment for SARS‐CoV‐2 infection. This article is protected by copyright. All rights reserved

negative: Comprehensive codon usage analysis of porcine deltacoronavirus Abstract Porcine deltacoronavirus (PDCoV) is a newly identified coronavirus of pigs that was first reported in Hong Kong in 2012. Since then, many PDCoV isolates have been identified worldwide. In this study, we analyzed the codon usage pattern of the S gene using complete coding sequences and complete PDCoV genomes to gain a deeper understanding of their genetic relationships and evolutionary history. We found that during evolution three groups evolved with a relatively low codon usage bias (effective number of codons (ENC) of 52). The factors driving bias were complex. However, the primary element influencing the codon bias of PDCoVs was natural selection. Our results revealed that different natural environments may have a significant impact on the genetic characteristics of the strains. In the future, more epidemiological surveys are required to examine the factors that resulted in the emergence and outbreak of this virus.

negative: Applying Lexical Link Analysis to Discover Insights from Public Information on COVID-19 SARS-Cov-2, the deadly and novel virus, which has caused a worldwide pandemic and drastic loss of human lives and economic activities. An open data set called the COVID-19 Open Research Dataset or CORD-19 contains large set full text scientific literature on SARS-CoV-2. The Next Strain consists of a database of SARS-CoV-2 viral genomes from since 12/3/2019. We applied an unique information mining method named lexical link analysis (LLA) to answer the call to action and help the science community answer high-priority scientific questions related to SARS-CoV-2. We first text-mined the CORD-19. We also data-mined the next strain database. Finally, we linked two databases. The linked databases and information can be used to discover the insights and help the research community to address high-priority questions related to the SARS-CoV-2’s genetics, tests, and prevention. Significance Statement In this paper, we show how to apply an unique information mining method lexical link analysis (LLA) to link unstructured (CORD-19) and structured (Next Strain) data sets to relevant publications, integrate text and data mining into a single platform to discover the insights that can be visualized, and validated to answer the high-priority questions of genetics, incubation, treatment, symptoms, and prevention of COVID-19.

negative: Multicluster nosocomial outbreak of parainfluenza virus type 3 infection in a pediatric oncohematology unit: a phylogenetic study. BACKGROUND Human parainfluenza virus type 3 (hPIV-3) has been reported to cause nosocomial outbreaks of respiratory infection, in particular among hematopoietic stem cell transplantation recipients. DESIGN AND METHODS From September 2007 through January 2008 several episodes of hPIV-3 infection were observed among young patients followed at the Oncohematology Unit (OHU) or other units of the Pediatrics Department. In 32 young patients (median age 3.5 years, range 21 days-27 years), hPIV-3 infection was diagnosed by direct fluorescent antibody staining of cells from respiratory secretions, and virus quantified by real-time RT-PCR in nasopharyngeal aspirates or bronchoalveolar lavage samples. In addition, the epidemiologic relatedness of hPIV-3 strains was investigated by sequencing two variable regions of the hemagglutinin-neuraminidase gene (nt 1-569 and nt 762-1239). RESULTS Of the 32 hPIV-3-positive patients, 19 were hematopoietic stem cell transplantation recipients, 8 had hematologic malignancies, and 5 were immunocompetent children. Sixteen patients had upper, and 16 lower respiratory tract infection. All patients but one had high viral load in nasopharyngeal aspirates (>1.0x10(6) RNA copies/mL). One patient died from respiratory failure with a high viral load in bronchoalveolar lavage. Phylogenetic analysis showed that 16/32 strains were identical. Besides this major cluster, three other clusters were identified, each one defining a smaller outbreak. CONCLUSIONS Phylogenetic analysis allows identification of the role of a single or multiple hPIV-3 strains in the person-to-person transmission within an outbreak occurring in clinical units.

negative: A Snapshot of the Global Race for Vaccines Targeting SARS-CoV-2 and the COVID-19 Pandemic A novel coronavirus SARS-CoV-2 causing Coronavirus disease 2019 (COVID-19) has entered the human population and has spread rapidly around the world in the first half of 2020 causing a global pandemic. The virus uses its spike glycoprotein receptor-binding domain to interact with host cell angiotensin-converting enzyme 2 (ACE2) sites to initiate a cascade of events that culminate in severe acute respiratory syndrome in some individuals. In efforts to curtail viral spread, authorities initiated far-reaching lockdowns that have disrupted global economies. The scientific and medical communities are mounting serious efforts to limit this pandemic and subsequent waves of viral spread by developing preventative vaccines and repurposing existing drugs as potential therapies. In this review, we focus on the latest developments in COVID-19 vaccine development, including results of the first Phase I clinical trials and describe a number of the early candidates that are emerging in the field. We seek to provide a balanced coverage of the seven main platforms used in vaccine development that will lead to a desired target product profile for the “ideal” vaccine. Using tales of past vaccine discovery efforts that have taken many years or that have failed, we temper over exuberant enthusiasm with cautious optimism that the global medical community will reach the elusive target to treat COVID-19 and end the pandemic.

negative: Origins and Evolution of the Global RNA Virome Viruses with RNA genomes dominate the eukaryotic virome, reaching enormous diversity in animals and plants. The recent advances of metaviromics prompted us to perform a detailed phylogenomic reconstruction of the evolution of the dramatically expanded global RNA virome. The only universal gene among RNA viruses is the gene encoding the RNA-dependent RNA polymerase (RdRp). We developed an iterative computational procedure that alternates the RdRp phylogenetic tree construction with refinement of the underlying multiple-sequence alignments. The resulting tree encompasses 4,617 RNA virus RdRps and consists of 5 major branches; 2 of the branches include positive-sense RNA viruses, 1 is a mix of positive-sense (+) RNA and double-stranded RNA (dsRNA) viruses, and 2 consist of dsRNA and negative-sense (−) RNA viruses, respectively. This tree topology implies that dsRNA viruses evolved from +RNA viruses on at least two independent occasions, whereas −RNA viruses evolved from dsRNA viruses. Reconstruction of RNA virus evolution using the RdRp tree as the scaffold suggests that the last common ancestors of the major branches of +RNA viruses encoded only the RdRp and a single jelly-roll capsid protein. Subsequent evolution involved independent capture of additional genes, in particular, those encoding distinct RNA helicases, enabling replication of larger RNA genomes and facilitating virus genome expression and virus-host interactions. Phylogenomic analysis reveals extensive gene module exchange among diverse viruses and horizontal virus transfer between distantly related hosts. Although the network of evolutionary relationships within the RNA virome is bound to further expand, the present results call for a thorough reevaluation of the RNA virus taxonomy.

negative: Phylogenetic analysis of porcine reproductive and respiratory syndrome virus isolates from Northern Ireland. To investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in Northern Ireland, the ORF5 gene from nine field isolates was sequenced and phylogenetically analysed. The results revealed relatively high diversity amongst isolates, with 87.6-92.2% identity between farms at the nucleotide level and 84.1-93.5% identity at the protein level. Phylogenetic analysis confirmed that all nine isolates belonged to the European (type 1) genotype and formed a cluster within the subtype 1 subgroup. This study provides the first report on PRRSV isolate diversity in Northern Ireland.

negative: Genotype and phenotype of COVID-19: Their roles in pathogenesis Abstract COVID-19 is a novel coronavirus with an outbreak of unusual viral pneumonia in Wuhan, China, and then pandemic. Based on its phylogenetic relationships and genomic structures the COVID-19 belongs to genera Betacoronavirus. Human Betacoronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV) have many similarities, but also have differences in their genomic and phenotypic structure that can influence their pathogenesis. COVID-19 is containing single-stranded (positive-sense) RNA associated with a nucleoprotein within a capsid comprised of matrix protein. A typical CoV contains at least six ORFs in its genome. All the structural and accessory proteins are translated from the sgRNAs of CoVs. Four main structural proteins are encoded by ORFs 10, 11 on the one-third of the genome near the 3′-terminus. The genetic and phenotypic structure of COVID-19 in pathogenesis is important. This article highlights the most important of these features compared to other Betacoronaviruses.

negative: At the Heart of the Matter: Unmasking and Addressing COVID-19's Toll on Diverse Populations

negative: Genomic evidence of homologous recombination in spring viremia of carp virus: a negatively single stranded RNA virus. A new strain of spring viraemia of carp virus, denominated SVCV-265, was isolated from an ornamental common carp (Cyprinus carpio) in Shanghai, China, 2013. The isolate could produce obvious cytopathic effects on EPC cells, while was shown to be of low virulence for juvenile koi. Complete genome sequencing revealed the genome of the SVCV-265 strain is 11,029 nucleotides in length and phylogenetic analysis showed the isolate was clustered within Asia clade but was divergent from Chinese A1, A2 and BJ0505-2 strains. Previous report indicated that the G and P gene of SVCV shared similar topologies of evolutionary trees. In this study, phylogenetic analysis based on the P gene sequences showed the SVCV-265 was clustered into Iai subgroup and divergent from Chinese isolates A1, A2 and BJ0505-2, which were clustered into Iaii group. However, sequence alignment of the G gene showed the SVCV-265 has a close relationship with A1, A2 and BJ0505-2 isolates. Recombination analysis of all the whole sequences of SVCV available revealed isolates A2 and BJ0505-2 were likely the homologous recombination descendants of the A1 and SVCV-265. The crossover regions were located between 3845-6387nt for A2 and 3573-6444 nt for BJ0505-2, respectively. Phylogenetic analysis of the crossover region further confirmed these findings. This current study describes the molecular characterization of the new isolate SVCV-265 from China and is the first report of homologous recombination in SVCV.

negative: Evolution and epidemic spread of SARS-CoV-2 in Brazil Brazil currently has one of the fastest growing SARS-CoV-2 epidemics in the world. Due to limited available data, assessments of the impact of non-pharmaceutical interventions (NPIs) on virus transmission and epidemic spread remain challenging. We investigate the impact of NPIs in Brazil using epidemiological, mobility and genomic data. Mobility-driven transmission models for Sao Paulo and Rio de Janeiro cities show that the reproduction number (Rt) reached below 1 following NPIs but slowly increased to values between 1 to 1.3 (1.0 - -1.6). Genome sequencing of 427 new genomes and analysis of a geographically representative genomic dataset from 21 of the 27 Brazilian states identified >100 international introductions of SARS-CoV-2 in Brazil. We estimate that three clades introduced from Europe emerged between 22 and 27 February 2020, and were already well-established before the implementation of NPIs and travel bans. During this first phase of the epidemic establishment of SARS-CoV-2 in Brazil, we find that the virus spread mostly locally and within-state borders. Despite sharp decreases in national air travel during this period, we detected a 25% increase in the average distance travelled by air passengers during this time period. This coincided with the spread of SARS-CoV-2 from large urban centers to the rest of the country. In conclusion, our results shed light on the role of large and highly connected populated centres in the rapid ignition and establishment of SARS-CoV-2, and provide evidence that current interventions remain insufficient to keep virus transmission under control in Brazil.

negative: Genomic epidemiology, evolution, and transmission dynamics of porcine deltacoronavirus. The emergence of severe acute respiratory syndrome virus (SARS)-CoV-2 has shown once again that coronaviruses (CoVs) in animals are potential sources for epidemics in humans. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen of swine with a worldwide distribution. Here, we implemented and described an approach to analyze the epidemiology of PDCoV following its emergence in the pig population. We performed an integrated analysis of full genome sequence data from 21 newly-sequenced viruses, along with comprehensive epidemiological surveillance data collected globally over the last 15 years. We found four distinct phylogenetic lineages of PDCoV, which differ in their geographic circulation patterns. Interestingly, we identified more frequent intra- and inter-lineage recombination and higher virus genetic diversity in the Chinese lineages compared with the US lineage where pigs are raised in different farming systems and ecological environments. Most recombination breakpoints are located in the ORF1ab gene rather than in genes encoding structural proteins. We also identified five amino acids under positive selection in the spike protein suggesting a role for adaptive evolution. According to structural mapping, three positively selected sites are located in the N-terminal domain of the S1 subunit, which is the most likely involved in binding to a carbohydrate receptor, while the other two are located in or near the fusion peptide of the S2 subunit and thus might affect membrane fusion. Finally, our phylogeographic investigations highlighted notable South-North transmission as well as frequent long-distance dispersal events in China that could implicate human-mediated transmission. Our findings provide new insights into the evolution and dispersal of PDCoV that contribute to our understanding of the critical factors involved in coronaviruses emergence. SIGNIFICANCE Coronaviruses (CoVs) exhibit a pronounced propensity for interspecies transmission as illustrated by important emerging viruses in humans such as severe acute respiratory syndrome virus (SARS)-CoV and Middle East respiratory syndrome virus (MERS)-CoV, as well as the recent SARS-CoV-2 that is causing a major human pandemic. A better understanding of the evolutionary process leading to viral emergence may help unravel the principles of cross-species transmission and therefore assist in preventing future introductions into new host populations. Here, we examined the evolution and spread of an emerging coronavirus that likely transmitted from birds to pigs and caused sustained epidemics in Asia and the United States. We integrated viral full genome sequence analysis and comprehensive surveillance data to examine the evolution of emerging porcine deltacoronavirus (PDCoV), and in particular the factors driving ongoing circulation and recent cases in China. We found evidence for adaptive evolution of this emerging virus in the absence of vaccine immune pressure, with important amino acids under positive selection, as well as frequent inter- and intra-lineage recombination events in the Asian lineages. Breeding patterns and ecological environment could play an important role in swine coronavirus evolution, leading to differences in recombination and population diversity. Using phylogeographic approaches, we further shed light on the dispersal history and dynamics of the China lineage of PDCoV. Our study provides a deeper understanding of the geographic dispersal of genetic lineages of PDCoV in China and suggests that southern China acted as a source of transmission. Modeling how the virus spread in China will allow the prioritization of areas for swine coronavirus surveillance in the future. These findings have important implications for preventing the emergence of coronaviruses in livestock and for controlling their spread in Asian countries.

negative: Phylogenetic host specificity and understanding parasite sharing in primates. Understanding how parasites are transmitted to new species is of great importance for human health, agriculture and conservation. However, it is still unclear why some parasites are shared by many species, while others have only one host. Using a new measure of 'phylogenetic host specificity', we find that most primate parasites with more than one host are phylogenetic generalists, infecting less closely related primates than expected. Evolutionary models suggest that phylogenetic host generalism is driven by a mixture of host-parasite cospeciation and lower rates of parasite extinction. We also show that phylogenetic relatedness is important in most analyses, but fails to fully explain patterns of parasite sharing among primates. Host ecology and geographical distribution emerged as key additional factors that influence contacts among hosts to facilitate sharing. Greater understanding of these factors is therefore crucial to improve our ability to predict future infectious disease risks.

negative: Evaluation of a Phylogenetic Marker Based on Genomic Segment B of Infectious Bursal Disease Virus: Facilitating a Feasible Incorporation of this Segment to the Molecular Epidemiology Studies for this Viral Agent BACKGROUND: Infectious bursal disease (IBD) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. The current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment B. This marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent IBD virus (vvIBDV) strains worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Sequences of the segment B gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank Database; Cuban sequences were obtained in the current work. A phylogenetic marker named B-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. This last parameter is based on the ability of B-marker to reconstruct the same topology as the complete segment B of the viral genome. From the results obtained from B-marker, demographic history for both main lineages of IBDV regarding segment B was performed by Bayesian skyline plot analysis. Phylogenetic analysis for both segments of IBDV genome was also performed, revealing the presence of a natural reassortant strain with segment A from vvIBDV strains and segment B from non-vvIBDV strains within Cuban IBDV population. CONCLUSIONS/SIGNIFICANCE: This study contributes to a better understanding of the emergence of vvIBDV strains, describing molecular epidemiology of IBDV using the state-of-the-art methodology concerning phylogenetic reconstruction. This study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvIBDV strains. Therefore, it highlights the need to obtain information about both genome segments of IBDV for molecular epidemiology studies.

negative: Use of whole-genome sequencing in the molecular investigation of care-associated HCoV-OC43 infections in a hematopoietic stem cell transplant unit Abstract Background While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV−OC43 infections have rarely been investigated as healthcare-associated infections in this population. Objectives In this report, HCoV−OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. Study design Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient’s sequences. Amino acids substitutions were analysed. Results Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. Conclusions HCoV−OC43 may be implicated in healthcare-associated infections.

negative: Genotyping and phylogenetic analysis of canine parvovirus circulating in Egypt AIM: This study aimed to detect and characterize current genotypes of canine parvovirus (CPV) in Egypt during 2018. MATERIALS AND METHODS: A total of 50 fecal swabs were collected from clinically infected domestic dogs of 2-5 months of age, suspected to suffer from CPV infection, from Cairo and Giza Governorates. The samples were subjected to qualitative antigen detection using the rapid test, followed by isolation on Madin-Darby Canine Kidney (MDCK) cells, molecular characterization with partial amplification of VP2 gene using polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis. RESULTS: Out of 50 fecal samples, 20 samples were positive (40%) by Rapid CPV/canine coronavirus Ag Test Kit. These positive samples were cultured successfully on MDCK cells. Nine randomly chosen samples out of 30 apparently negative samples were amplified using PCR with primers Hfor and Hrev to yield a typical 630 bp fragment. Then, six randomly chosen samples out of nine were amplified using PCR with primers Pbs and Pbas to yield a typical 427 bp fragment. Sequencing, BLAST analysis and assembly of the two fragments (630 bp and 427 bp) to produce 912 bp fragments, in the six samples, revealed two serotypes CPV-2b and CPV-2c. The obtained strains were submitted to GenBank and given accession numbers MK642272, MK642273, MK642274, MK642275, MK642276, and MK642277. Phylogenetic analysis of the Egyptian strains serotype 2b illustrated that they were closely related to Thailand strains (accession numbers KP715709, KP715694, KP715701, and KP715700); while Egyptian strains serotype 2c was closely related to Thailand strains (accession numbers MH711894 and MH711902), Taiwanese strain (KU244254), Chinese strain (MF467242), and Vietnamese strain (accession number LC216910). CONCLUSION: The current research recommends further epidemiological studies to assess the extent of the occurrence of different serotypes of CPV in Egypt and the efficiency of imported and locally produced vaccines in protection against CPV infection.

negative: Spike-based phylogenetically defined clades within the Alphacoronavirus 1 species Taxonomic classification for the Coronaviridae can be challenging, due to the wide host tropism and highly variable genome of the viruses in this Family. Within the Alphacoronavirus genus, there is a single species Alphacoronavirus 1 that encompasses several biologically distinct viruses of distinct animal species. Here, we carried out phylogenetic analysis of members of the Alphacoronavirus genus, focusing on the viral spike gene, which is a primary driver of viral tropism and pathogenesis. We identify two distinct clades (A and B) within the Alphacoronavirus 1 species. Alphacoronavirus 1 clade A encompasses serotype I FCoV and CCoV, and Alphacoronavirus 1 clade B, encompasses serotype II FCoV and CCoV and TGEV-like viruses. We propose this clade designation, along with the newly proposed Alphacoronavirus 2 species, as an improved way to classify the diverse Alphacoronavirus genus.

negative: SARS-CoV-2: an Emerging Coronavirus that Causes a Global Threat An ongoing outbreak of pneumonia caused by a novel coronavirus, currently designated as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), was reported recently. However, as SARS-CoV-2 is an emerging virus, we know little about it. In this review, we summarize the key events occurred during the early stage of SARS-CoV-2 outbreak, the basic characteristics of the pathogen, the signs and symptoms of the infected patients as well as the possible transmission pathways of the virus. Furthermore, we also review the current knowledge on the origin and evolution of the SARS-CoV-2. We highlight bats as the potential natural reservoir and pangolins as the possible intermediate host of the virus, but their roles are waiting for further investigation. Finally, the advances in the development of chemotherapeutic options are also briefly summarized.

negative: SARS-CoV-2 pathophysiology and assessment of coronaviruses in CNS diseases with a focus on therapeutic targets The novel Coronavirus disease of 2019 (nCOV-19) is a viral outbreak noted first in Wuhan, China. This disease is caused by Severe Acute Respiratory Syndrome (SARS) Coronavirus (CoV)-2. In the past, other members of the coronavirus family, such as SARS and Middle East Respiratory Syndrome (MERS), have made an impact in China and the Arabian peninsula respectively. Both SARS and COVID-19 share similar symptoms such as fever, cough, and difficulty in breathing that can become fatal in later stages. However, SARS and MERS infections were epidemic diseases constrained to limited regions. By March 2020 the SARS-CoV-2 had spread across the globe and on March 11th, 2020 the World Health Organization (WHO) declared COVID-19 as pandemic disease. In severe SARS-CoV-2 infection, many patients succumbed to pneumonia. Higher rates of deaths were seen in older patients who had co-morbidities such as diabetes mellitus, hypertension, cardiovascular disease (CVD), and dementia. In this review paper, we discuss the effect of SARS-CoV-2 on CNS diseases, such as Alzheimer's-like dementia, and diabetes mellitus. We also focus on the virus genome, pathophysiology, theranostics, and autophagy mechanisms. We will assess the multiorgan failure reported in advanced stages of SARS-CoV-2 infection. Our paper will provide mechanistic clues and therapeutic targets for physicians and investigators to combat COVID-19.

negative: ELM—the database of eukaryotic linear motifs Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instances.

negative: A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

negative: Sarbecovirus comparative genomics elucidates gene content of SARS-CoV-2 and functional impact of COVID-19 pandemic mutations Despite its overwhelming clinical importance for understanding and mitigating the COVID-19 pandemic, the protein-coding gene content of the SARS-CoV-2 genome remains unresolved, with the function and even protein-coding status of many hypothetical proteins unknown and often conflicting among different annotations, thus hindering efforts for systematic dissection of its biology and the impact of recent mutations. Comparative genomics is a powerful approach for distinguishing protein-coding versus non-coding functional elements, based on their characteristic patterns of change, which we previously used to annotate protein-coding genes in human, fly, and other species. Here, we use comparative genomics to provide a high-confidence set of SARS-CoV-2 protein-coding genes, to characterize their protein-level and nucleotide-level evolutionary constraint, and to interpret the functional implications for SARS-CoV-2 mutations acquired during the current pandemic. We select 44 complete Sarbecovirus genomes at evolutionary distances well-suited for protein-coding and non-coding element identification, create whole-genome alignments spanning all named and putative genes, and quantify their protein-coding evolutionary signatures using PhyloCSF and their overlapping constraint using FRESCo. We find strong protein-coding signatures for all named genes and for hypothetical ORFs 3a, 6, 7a, 7b, and 8, indicating protein-coding roles, and provide strong evidence of protein-coding status for a recently-proposed alternate-frame novel ORF within 3a. By contrast, ORF10 shows no protein-coding signatures but shows unusually-high nucleotide-level constraint, indicating it has important but non-coding functions, and ORF14 and SARS-CoV-1 ORF3b, which overlap other genes, lack evolutionary signatures expected for dual-coding regions, indicating they do not produce functional proteins. ORF9b has ambiguous protein-coding signatures, preventing us from resolving its protein-coding status. ORF8 shows extremely fast nucleotide-level evolution, lacks a known function, and was deactivated in SARS-CoV-1, but shows clear signatures indicating protein-coding function worthy of further investigation given its rapid evolution and potential role in replication. SARS-CoV-2 mutations are preferentially excluded from evolutionarily-constrained amino acid residues and synonymously-constrained nucleotides, indicating purifying constraint acting at both coding and non-coding levels. In contrast, we find a conserved region in the nucleocapsid that is enriched for recent mutations, which could indicate a selective signal, and find that several spike-protein mutations previously identified as candidates for increased transmission and several mutations in isolates found to generate higher viral load in-vitro disrupt otherwise-perfectly-conserved amino-acids, consistent with adaptations for human-to-human transmission.

negative: Disparate temperature-dependent virus – host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium The human conductive respiratory tract spans a long anatomical distance and represents an important barrier to constrain invading respiratory pathogens. The disparate ambient temperatures found in the upper and lower respiratory tract have been demonstrated to influence the replication kinetics of common cold viruses as well as the associated host responses. Here, we employed the human airway epithelial cell (hAEC) culture model to investigate the impact of ambient temperatures found in the upper and lower respiratory tract, 33°C and 37°C, respectively, on the viral replication kinetics and host innate immune response dynamics during SARS-CoV-2 and SARS-CoV infections. Strikingly, SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs than SARS-CoV. Time-resolved transcriptome analysis highlighted a temperature-dependent induction of IFN-mediated antiviral response, whose amplitude inversely correlated with the replication kinetic efficiencies of both SARS-CoV-2 and SARS-CoV at temperatures found in the upper and lower respiratory tract. Altogether, these data reflect clinical features of SARS-CoV-2 and SARS-CoV and subsequently, their associated human-to-human transmission efficiencies. They provide crucial insights of the profound impact of ambient temperatures on viral replication and associated pivotal virus - host interaction dynamics. This knowledge can be exploited for the development of novel intervention strategies against SARS-CoV-2.

negative: A fast and simple one-step duplex PCR assay for canine distemper virus (CDV) and canine coronavirus (CCoV) detection The one-step polymerase chain reaction (one-step PCR) detection assay is an innovative PCR detection method, eliminating nucleic acid extraction steps, in which samples can be directly added to PCR reagents for testing. For simultaneous detection of CDV and CCoV, a sensitive and specific one-step duplex PCR (one-step dPCR) assay was developed with two pairs of primers that were designed based on H and M gene sequences of CDV and CCoV, respectively. The one-step dPCR with optimized detection conditions has high specificity and sensitivity; independent sequencing assays further verified these results.

negative: SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability The emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.

negative: Excavating SARS‐coronavirus 2 genome for epitope‐based subunit vaccine synthesis using immunoinformatics approach Since the outbreak of severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) in December 2019 in China, there has been an upsurge in the number of deaths and infected individuals throughout the world, thereby leading to the World Health Organization declaration of a pandemic. Since no specific therapy is currently available for the same, the present study was aimed to explore the SARS‐CoV‐2 genome for the identification of immunogenic regions using immunoinformatics approach. A series of computational tools were applied in a systematic way to identify the epitopes that could be utilized in vaccine development. The screened‐out epitopes were passed through several immune filters, such as promiscuousity, conservancy, antigenicity, nonallergenicity, population coverage, nonhomologous to human proteins, and affinity with human leukocyte antigen alleles, to screen out the best possible ones. Further, a construct comprising 11 CD4, 12 CD8, 3 B cell, and 3 interferon‐γ epitopes, along with an adjuvant β‐defensin, was designed in silico, resulting in the formation of a multiepitope vaccine. The in silico immune simulation and population coverage analysis of the vaccine sequence showed its capacity to elicit cellular, humoral, and innate immune cells and to cover up a worldwide population of more than 97%. Further, the interaction analysis of the vaccine construct with Toll‐like receptor 3 (immune receptor) was carried out by docking and dynamics simulations, revealing high affinity, constancy, and pliability between the two. The overall findings suggest that the vaccine may be highly effective, and is therefore required to be tested in the lab settings to evaluate its efficacy.

negative: A further analysis of Manning and Fink (2020)

negative: Transmission and evolutionary dynamics of human coronavirus OC43 strains in coastal Kenya investigated by partial spike sequence analysis, 2015–16 Human coronavirus OC43 (HCoV-OC43) is a major contributor to seasonal outbreaks of acute respiratory illness (ARI). The origins of locally circulating HCoV-OC43 strains and characteristics of their genetic diversity are unknown for most settings despite significance to effective HCoV control strategies. Between December 2015 and June 2016, we undertook ARI surveillance in coastal Kenya in nine outpatients and one inpatient health facility (HF). Ninety-two patient samples tested HCoV-OC43 positive and forty (43.5%) were successfully sequenced in spike (S) gene region (2,864 long, ∼70%). Phylogenetic analysis confirmed co-circulation of two distinct HCoV-OC43 clades that closely clustered with genotype G (n = 34, 85%) and genotype H (n = 6, 15%) reference strains. Local viruses within the same clade displayed low genetic diversity yielding identical sequences in multiple HF. Furthermore, the newly sequenced Kenyan viruses showed close phylogenetic relationship to other contemporaneous sampled strains (2015–16) including those originating from distant places (e.g. USA and China). Using a genetic similarity threshold of 99.1 per cent at nucleotide level, the HCoV-OC43 strains sampled globally between 1967 and 2019 fell into nine sequence clusters. Notably, some of these clusters appeared to have become extinct, or occurred only sporadically in a few geographical areas while others persisted globally for multiple years. In conclusion, we found that HCoV-OC43 strains spread rapidly both locally and across the globe with limited genetic evolution in the spike gene. Full-genome sequences that are spatio-temporally representative are required to advance understanding of the transmission pathways of this important human respiratory pathogen.

negative: Bio-informed Protein Sequence Generation for Multi-class Virus Mutation Prediction Viral pandemics are emerging as a serious global threat to public health, like the recent outbreak of COVID-19. Viruses, especially those belonging to a large family of +ssRNA viruses, have a high possibility of mutating by inserting, deleting, or substituting one or multiple genome segments. It is of great importance for human health worldwide to predict the possible virus mutations, which can effectively avoid the potential second outbreak. In this work, we develop a GAN-based multi-class protein sequence generative model, named ProteinSeqGAN. Given the viral species, the generator is modeled on RNNs to predict the corresponding antigen epitope sequences synthesized by viral genomes. Additionally, a Graphical Protein Autoencoder (GProAE) built upon VAE is proposed to featurize proteins bioinformatically. GProAE, as a multi-class discriminator, also learns to evaluate the goodness of protein sequences and predict the corresponding viral species. Further experiments show that our ProteinSeqGAN model can generate valid antigen protein sequences from both bioinformatics and statistics perspectives, which can be promising predictions of virus mutations.

negative: Unrevealing sequence and structural features of novel coronavirus using in silico approaches: The main protease as molecular target Direct-acting antivirals are effective tools to control viral infections. SARS-CoV-2 is a coronavirus associated with the epidemiological outbreak in late 2019. Previous reports showed that HIV-1 protease inhibitors could block SARS-CoV main protease. Based on that and using an in silico approach, we evaluated SARS-CoV-2 main protease as a target for HIV-1 protease inhibitors to reveal the structural features related to their antiviral effect. Our results showed that several HIV inhibitors such as lopinavir, ritonavir, and saquinavir produce strong interaction with the active site of SARS-CoV-2 main protease. Furthermore, broad library protease inhibitors obtained from PubChem and ZINC (www.zinc.docking.org) were evaluated. Our analysis revealed 20 compounds that could be clustered into three groups based on their chemical features. Then, these structures could serve as leading compounds to develop a series of derivatives optimizing their activity against SARS-CoV-2 and other coronaviruses. Altogether, the results presented in this work contribute to gain a deep understanding of the molecular pharmacology of SARS-CoV-2 treatment and validate the use of protease inhibitors against SARS-CoV-2.

negative: Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1(A) and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on “up” RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3–53/VH3–66 and similarity to a SARS-CoV VH3–30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

negative: Automated analysis of phylogenetic clusters BACKGROUND: As sequence data sets used for the investigation of pathogen transmission patterns increase in size, automated tools and standardized methods for cluster analysis have become necessary. We have developed an automated Cluster Picker which identifies monophyletic clades meeting user-input criteria for bootstrap support and maximum genetic distance within large phylogenetic trees. A second tool, the Cluster Matcher, automates the process of linking genetic data to epidemiological or clinical data, and matches clusters between runs of the Cluster Picker. RESULTS: We explore the effect of different bootstrap and genetic distance thresholds on clusters identified in a data set of publicly available HIV sequences, and compare these results to those of a previously published tool for cluster identification. To demonstrate their utility, we then use the Cluster Picker and Cluster Matcher together to investigate how clusters in the data set changed over time. We find that clusters containing sequences from more than one UK location at the first time point (multiple origin) were significantly more likely to grow than those representing only a single location. CONCLUSIONS: The Cluster Picker and Cluster Matcher can rapidly process phylogenetic trees containing tens of thousands of sequences. Together these tools will facilitate comparisons of pathogen transmission dynamics between studies and countries.

negative: Tracking the onset date of the community spread of SARS-CoV-2 in Western Countries The SARS-CoV-2 rapidly spread around the world during 2020, but the precise time in which the virus began to spread locally is currently unknown for most countries. Here, we estimate the probable onset date of the community spread of SARS-CoV-2 from the cumulative number of deaths reported during the early stage of the epidemic in Western Europe and the Americas. Our results support that SARS-CoV-2 probably started to spread locally in all western countries analyzed between the middle of January and early February 2020, thus long before community transmission was officially recognized and control measures were implemented.

negative: Molecular characterization of pathogenic 4/91-like and QX-like infectious bronchitis virus infecting commercial poultry farms in Indonesia BACKGROUND AND AIM: Existing data on the characteristics of infectious bronchitis virus (IBV) gathered throughout Indonesia have been recognized to indicate variants similar to globally distributed vaccine strains. Despite past and current intensive vaccination programs, IBV infections in the country’s poultry industry have not been effectively controlled. Therefore, this study aimed to investigate the genotype of several isolates based on partial S1 gene sequences. In particular, the investigation is directed to focus on layer chickens in actively vaccinated farms indicating IBV symptoms. MATERIALS AND METHODS: Samples were isolated from ten different layer chicken flocks experiencing respiratory problem, drops in egg production, and a “penguin-like” stance, which were collected from commercial poultry farms in Central Java and Yogyakarta regions, Indonesia, within the periods of 2012-2018. Fragment of the S1 gene of IBV sampled from actively vaccinated commercial poultry farms was amplified using primer 5’-aca tgg taa ttt ttc aga tgg-3’ (forward) and 5’-cag att gct tac aac cac c-3’ (reverse) with the length of polymerase chain reaction (PCR) product at 383 bp. The sequence of samples was then compared with the sequence of reference S1 gene nucleotides of IBV from NCBI GenBank database. The amino acid analysis and multiple alignment sequence were conducted using Mega X. RESULTS: During necropsy, enlargement of the oviduct and swollen kidney were observed. Reverse transcription-PCR diagnosis of their 383 bp S1 gene showed that all samples were IBV positive. Phylogenetic analysis of the S1 gene discovered seven samples to be clustered as 4/91-like strains. Meanwhile, the remaining three samples were grouped in QX-like strain cluster. CONCLUSION: This study is a pioneering report providing molecular evidence of pathogenic QX-like and 4/91-like strains circulating in Indonesia. Findings discovered, in this study, strongly suggested the importance of improving protections by available IBV vaccines through updated circulating strain clusters. It is critical to ensure the delivery of an effective control measurement of and vaccination protocols against IBV infections in the country’s commercial poultry industry in particular and worldwide in general.

negative: Mutated COVID-19 may foretell a great risk for mankind in the future Coronavirus disease 2019 SARS-CoV-2 (COVID-19) is a zoonotic virus causing a variety of severe respiratory diseases. SARS-CoV-2 is closest to SARS-CoV and MERS-CoV in structure. The high prevalence of COVID-19 is a result of a lack of symptoms at onset. Our study aimed to present an overview of the virus in terms of structure, epidemiology, symptoms, treatment and prevention. Whole genome sequences and some viral proteins were investigated to determine gaps and changes in alternation of nucleotides and amino acid sequences. We evaluate 11 complete genome sequences of different coronaviruses using BAST and MAFFT software. We also selected seven types of structural proteins. We conclude that COVID-19 might produce new mutations, specifically in glycoproteins, so caution and complete preparation by health authorities is required.

negative: ACE2 coding variants in different populations and their potential impact on SARS-CoV-2 binding affinity The susceptibility of different populations to the SARS-CoV-2 infection is not yet understood. A deeper analysis of the genomes of individuals from different populations might explain their risk for infection. In this study, a combined analysis of ACE2 coding variants in different populations and computational chemistry calculations are conducted in order to probe the potential effects of ACE2 coding variants on SARS-CoV-2/ACE2 binding affinity. Our study reveals novel interaction data on the variants and SARS-CoV-2. We could show that ACE2-K26R; which is more frequent in the Ashkenazi Jewish population decrease the electrostatic attraction between SARS-CoV-2 and ACE2. On the contrary, ACE2-I468V, R219C, K341R, D206G, G211R were found to increase the electrostatic attraction and increase the binding to SARS-CoV-2; ordered by the strength of binding from weakest to strongest. I468V, R219C, K341R, D206G and G211R were more frequent in East Asian, South Asian, African and African American, European and European and South Asian populations, respectively. SARS-CoV-2/ACE2 interface in the WT protein and corresponding variants is showed to be a dominated by van der Waals (vdW) interactions. All the mutations except K341R induce an increase in the vdW attractions between the ACE2 and the SARS-CoV-2. The largest increase of is observed for the R219C mutant.

negative: Molecular epidemiology, evolution and phylogeny of SARS coronavirus Abstract Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs (SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats [mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9]. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species.

negative: The ORF3a protein of SARS-CoV-2 induces apoptosis in cells

negative: The impact of super-spreaders in COVID-19: mapping genome variation worldwide The human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the major pandemic of the 21st century. We analyzed >4,700 SARS-CoV-2 genomes and associated meta-data retrieved from public repositories. SARS-CoV-2 sequences have a high sequence identity (>99.9%), which drops to >96% when compared to bat coronavirus. We built a mutation-annotated reference SARS-CoV-2 phylogeny with two main macro-haplogroups, A and B, both of Asian origin, and >160 sub-branches representing virus strains of variable geographical origins worldwide, revealing a uniform mutation occurrence along branches that could complicate the design of future vaccines. The root of SARS-CoV-2 genomes locates at the Chinese haplogroup B1, with a TMRCA dating to 12 November 2019 - thus matching epidemiological records. Sub-haplogroup A2a originates in China and represents the major non-Asian outbreak. Multiple founder effect episodes, most likely associated with super-spreader hosts, explain COVID-19 pandemic to a large extent.

negative: Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target

negative: SARS‐coronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS Background Angiotensin converting enzyme 2 (ACE2), a monocarboxylase that degrades angiotensin II to angiotensin 1–7, is also the functional receptor for severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) and is highly expressed in the lungs and heart. Patients with SARS also suffered from cardiac disease including arrhythmias, sudden cardiac death, and systolic and diastolic dysfunction. Materials and methods We studied mice infected with the human strain of the SARS‐CoV and encephalomyocarditis virus and examined ACE2 mRNA and protein expression. Autopsy heart samples from patients who succumbed to the SARS crisis in Toronto (Canada) were used to investigate the impact of SARS on myocardial structure, inflammation and ACE2 protein expression. Results Pulmonary infection with the human SARS‐CoV in mice led to an ACE2‐dependent myocardial infection with a marked decrease in ACE2 expression confirming a critical role of ACE2 in mediating SARS‐CoV infection in the heart. The SARS‐CoV viral RNA was detected in 35% (7/20) of autopsied human heart samples obtained from patients who succumbed to the SARS crisis during the Toronto SARS outbreak. Macrophage‐specific staining showed a marked increase in macrophage infiltration with evidence of myocardial damage in patients who had SARS‐CoV in their hearts. The presence of SARS‐CoV in the heart was also associated with marked reductions in ACE2 protein expression. Conclusions Our data show that SARS‐CoV can mediate myocardial inflammation and damage associated with down‐regulation of myocardial ACE2 system, which may be responsible for the myocardial dysfunction and adverse cardiac outcomes in patients with SARS.

negative: COVID-19 Variants Database: A repository for Human SARS-CoV-2 Polymorphism Data COVID-19 is a newly communicable disease with a catastrophe outbreak that affects all over the world. We retrieved about 8,781 nucleotide fragments and complete genomes of SARS-CoV-2 reported from sixty-four countries. The CoV-2 reference genome was obtained from the National Genomics Data Center (NGDC), GISAID, and NCBI Genbank. All the sequences were aligned against reference genomes using Clustal Omega and variants were called using in-house built Python script. We intend to establish a user-friendly online resource to visualize the variants in the viral genome along with the Primer Infopedia. After analyzing and filtering the data globally, it was made available to the public. The detail of data available to the public includes mutations from 5688 SARS-CoV-2 sequences curated from 91 regions. This database incorporated 39920 mutations over 3990 unique positions. According to the translational impact, these mutations include 11829 synonymous mutations including 681 synonymous frameshifts and 21701 nonsynonymous mutations including 10 nonsynonymous frameshifts. Development of SARS-CoV-2 mutation genome browsers is a fundamental step obliging towards the virus surveillance, viral detection, and development of vaccine and therapeutic drugs. The SARS-COV-2 mutation browser is available at http://covid-19.dnageography.com.

negative: Reliable typing of MERS-CoV variants with a small genome fragment Abstract Background Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. Objective To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans. Study Design Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized. Results A reverse-transcription PCR assay for MERS-CoV targeting a 615bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9–32.2) yielded reliable sequence information in 14 samples. Conclusions We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location.

negative: Revealing COVID-19 Transmission by SARS-CoV-2 Genome Sequencing and Agent Based Modelling Community transmission of the new coronavirus SARS-CoV-2 is a major public health concern that remains difficult to assess. We present a genomic survey of SARS-CoV-2 from a during the first 10 weeks of COVID-19 activity in New South Wales, Australia. Transmission events were monitored prospectively during the critical period of implementation of national control measures. SARS-CoV-2 genomes were sequenced from 209 patients diagnosed with COVID-19 infection between January and March 2020. Only a quarter of cases appeared to be locally acquired and genomic-based estimates of local transmission rates were concordant with predictions from a computational agent-based model. This convergent assessment indicates that genome sequencing provides key information to inform public health action and has improved our understanding of the COVID-19 evolution from outbreak to epidemic.

negative: Structural analysis of the putative SARS-CoV-2 primase complex We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 β-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å(2) connects the nsp7 to nsp8 and a second one of approx. 950 Å(2) connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.

negative: Global genetic diversity patterns and transmissions of SARS-CoV-2 Background: Since it was firstly discovered in China, the SARS-CoV-2 epidemic has caused a substantial health emergency and economic stress in the world. However, the global genetic diversity and transmissions are still unclear. Methods: 3050 SARS-CoV-2 genome sequences were retrieved from GIASID database. After aligned by MAFFT, the mutation patterns were identified by phylogenetic tree analysis. Results: We detected 17 high frequency (>6%) mutations in the 3050 sequences. Based on these mutations, we classed the SARS-CoV-2 into four main groups and 10 subgroups. We found that group A was mainly presented in Asia, group B was primarily detected in North America, group C was prevailingly appeared in Asia and Oceania and group D was principally emerged in Europe and Africa. Additionally, the distribution of these groups was different in age, but was similar in gender. Group A, group B1 and group C2 were declined over time, inversely, group B2, group C3 and group D were rising. At last, we found two apparent expansion stages (late Jan-2020 and late Feb-2020 to early Mar-2020, respectively). Notably, most of groups are quickly expanding, especially group D. Conclusions: We classed the SARS-CoV-2 into four main groups and 10 subgroups based on different mutation patterns at first time. The distribution of the 10 subgroups was different in geography, time and age, but not in gender. Most of groups are rapidly expanding, especially group D. Therefore, we should attach importance to these genetic diversity patterns of SARS-CoV-2 and take more targeted measures to constrain its spread.

negative: CO.ME.T.A. -- covid-19 media textual analysis. A dashboard for media monitoring The focus of this paper is to trace how mass media, particularly newspapers, have addressed the issues about the containment of contagion or the explanation of epidemiological evolution. We propose an interactive dashboard: CO.ME.T.A.. During crises it is important to shape the best communication strategies in order to respond to critical situations. In this regard, it is important to monitor the information that mass media and social platforms convey. The dashboard allows to explore the mining of contents extracted and study the lexical structure that links the main discussion topics. The dashboard merges together four methods: text mining, sentiment analysis, textual network analysis and latent topic models. Results obtained on a subset of documents show not only a health-related semantic dimension, but it also extends to social-economic dimensions.

negative: Masks and thermometers: Paramount measures to stop the rapid spread of SARS-CoV-2 in the United States Abstract In the United States, there is currently an exponential growth for the COVID-19 cases. The US president’s coronavirus guidelines for Americans “30 Days to Slow The Spread” are necessary. To effectively curb the rapid spread of SARS-CoV-2, two more control measures masks and thermometers are strongly suggested to be included in the Guidelines.

negative: Molecular characterization of PL97-1, the first Korean isolate of the porcine reproductive and respiratory syndrome virus Abstract We determined the complete nucleotide and predicted amino acid sequence of the genomic RNA of PL97-1, the first Korean strain of porcine reproductive and respiratory syndrome virus (PRRSV), which was isolated from the serum of an infected pig in 1997. We found that the 15411-nucleotide genome of PL97-1 consisted of a 189-nucleotide 5′ noncoding region (NCR), a 15071-nucleotide protein-coding region, and a 151-nucleotide 3′NCR, followed by a poly (A) tail. The 5′-end of PL97-1 began with 1 ATG ACG TAT AGG12. Comparison of the PL97-1 genome with the 11 fully sequenced PRRSV genomes currently available revealed sequence divergence ranging from 0.3% (the VR-2332-derived vaccine MLV RespPRRS/Repro strain) to 38% (the Dutch Lelystad strain). To better understand the genetic relationships between these different strains, phylogenetic analyses were performed on the full-length PRRSV genomes. Significantly, the phylogenetic tree based on the ORF1b or ORF7 genes most closely resembled the tree based on the full-length genomes. Thus, these single genes will be the most useful in revealing the genetic relationships between the different strains relative to their geographical distribution. Extensive phylogenetic analyses using the ORF7 sequences of 111 PRRSV isolates available revealed that PL97-1 is most closely related to the North American genotype VR-2332, a VR-2332-derived vaccine strain, and Chinese BJ-4. It is distantly related to the European genotype Lelystad. This study provides the largest full-length genome phylogenetic analysis of PRRSV that has been published to date, and supports an earlier genetic grouping of the many temporally and geographically diverse PRRSV strains currently isolated.

negative: Structural basis of receptor recognition by SARS-CoV-2. A novel SARS-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans1,2. A key to tackling this epidemic is to understand the virus's receptor recognition mechanism, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor - human ACE2 (hACE2)3,4. Here we determined the crystal structure of the SARS-CoV-2 receptor-binding domain (RBD) (engineered to facilitate crystallization) in complex with hACE2. Compared with the SARS-CoV RBD, a hACE2-binding ridge in SARS-CoV-2 RBD takes a more compact conformation; moreover, several residue changes in SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD/hACE2 interface. These structural features of SARS-CoV-2 RBD enhance its hACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus closely related to SARS-CoV-2, also uses hACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in hACE2 recognition shed light on potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies targeting receptor recognition by SARS-CoV-2.

negative: Is SARS-CoV-2 Vertically Transmitted? At the end of 2019, in Wuhan (China), the onset of a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was observed. The disease, named COVID-19, has a wide spectrum of clinical presentations, ranging from asymptomatic or mild to critical, and for some patients the disease is even fatal. Apparently, being a child or being pregnant does not represent an additional risk for adverse outcomes. The purpose of this mini-review was to investigate what is in the scientific literature, so far, in regard to vertical transmission of SARS-CoV-2. Data were obtained independently by the two authors, who carried out a systematic search in the PubMed, Embase, LILACS, Cochrane, Scopus and SciELO databases using the Medical Subject Heading terms “coronavirus,” “COVID-19,” and “vertical transmission.” Few studies about the vertical transmission of SARS-CoV-2 are found in the literature. In all case reports and case series, the mothers' infection occurred in the third trimester of pregnancy, there were no maternal deaths, and most neonates had a favorable clinical course. The virus was not detected in the neonate nasopharyngeal swab samples at birth, in the placenta, in the umbilical cord, in the amniotic fluid, in the breast milk or in the maternal vaginal swab samples in any of these articles. Only three papers reported neonatal SARS-CoV-2 infection, but there is a bias that positive pharyngeal swab samples were collected at 36 h and on the 2nd, 4th, and 17th days of life. The possibility of intrauterine infection has been based mainly on the detection of IgM and IL-6 in the neonates' serum. In conclusion, to date, no convincing evidence has been found for vertical transmission of SARS-CoV-2.

negative: Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies Summary Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses and that characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

negative: Retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China Swine acute diarrhoea syndrome coronavirus (SADS‐CoV), a novel coronavirus, was first discovered in southern China in January 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. Here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of SADS‐CoV in pigs in China. Our results suggest that SADS‐CoV has emerged in China at least since August 2016. Meanwhile, we detected a prevalence of SADS‐CoV (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (PEDV) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of SADS‐CoV and PEDV occurred most frequently with the rate of 17.65%. We screened and obtained two new complete genomes, five N and five S genes of SADS‐CoV. Phylogenetic analysis based on these sequences revealed that all SADS‐CoV sequences in this study clustered with previously reported SADS‐CoV strains to form a well defined branch that grouped with the bat coronavirus HKU2 strains. This study is the first retrospective investigation for SADS‐CoV and provides the epidemiological information of this new virus in China, which highlights the urgency to develop effective measures to control SADS‐CoV.

negative: Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3 gene and the S1 gene BACKGROUND: Porcine epidemic diarrhea (PED) has increased in severity in China since 2010. To investigate further the infectivity, genetic diversity and molecular epidemiology of its causative agent, the porcine epidemic diarrhea virus (PEDV), we assessed 129 clinical samples, which were the intestinal tissue of piglets with severe diarrhea, from 17 cities in central China. Both the spike (S) glycoprotein (S1, 1–789 amino acids (aa)) and the full-length ORF3 gene of 21 representative field strains from 21 farms in 11 cities were sequenced and analysed. METHODS: PEDV was detected by reverse transcription-polymerase chain reaction (RT-PCR), and S1 and ORF3 sequences were processed by the Clustal W method via DNAMAN 8 software, and phylogenetic trees were constructed by the neighbor-joining method using MEGA 6 software. RESULTS: The prevalence of PEDV was 92.25% and was detected in 119 of 129 samples, with 94.03% (63 of 67) of pig farms harbouring the disease. According to the phylogenetic analysis of the S1 genes, our isolates all fell into group G2 (variants) and showed a close relationship to isolates from Chinese (HN1303, CH/ZMDZY/11 and AJ1102), Korean (AD01), American (MN, IA1, IA2 and 13–019349) sources, and these isolates differed genetically from other Chinese (LZC, CH/HNZZ/2011 and SD-M) and Korean (SM98) strains as well Japanese (83-P5 and MK) strains. In addition, our isolates differed from attenuated vaccine strains, CV777 (used in China) and DR13 (used in Korea). According to our derived amino acid sequence analysis, we detected one novel variant PEDV, viz: CH/HNLY, with 4-aa insertion/deletion (RSSS/T) at position 375 and 1-aa (D) deletion at position 430 compared to the CV777 attenuated strain. These mutations were located on the receptor binding domain. Our ORF3 gene analyses showed that the prevalent PEDV isolates were variants, and the isolated strains differed genetically from the vaccine strains. CONCLUSIONS: These findings illustrated the existence of genetic diversity among geographically distinct PEDV strains, and our study has provided an impetus to conduct further research on the PEDV receptor binding protein and on the new and efficacious vaccines design. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0646-8) contains supplementary material, which is available to authorized users.

negative: Structural landscape of the complete genomes of Dengue serotypes and other viral hemorrhagic fevers Background With more than 300 million potentially infected people every year, and with the expanded habitat of mosquitoes due to climate change, dengue cannot be considered anymore only a tropical disease. The RNA secondary structure is a functional characteristic of RNA viruses, and together with the accumulated high-throughput sequencing data could provide general insights towards understanding virus biology. Here, we profiled the RNA secondary structure of >7500 complete viral genomes from 11 different species of viral hemorrhagic fevers, including dengue serotypes, ebola, and yellow fever. Results We achieved hig prediction scores (AUC up to 0.85 with experimental data), and computed consensus secondary structure profiles using hundreds of structural in silico models. We observed that virulent viruses such as DENV-2 and ebola tend to be less structured than the other viruses. Furthermore, we observed virus-specific correlations between secondary structure and the number of interaction sites with human proteins, reaching a correlation of 0.89 in the case of zika. We demonstrate that the secondary structure and presence of protein-binding domains in the genomes can be used as intrinsic signature to further classify the viruses. We also used structural data to study the geographical distribution of dengue, finding a significant difference between DENV-3 from Asia and South-America, which could imply different evolutionary routes of this subtype. Conclusions Our massive computational analysis provided novel results regarding the secondary structure and the interaction with human proteins, not only for Dengue serotypes, but also for other viral hemorrhagic fevers. We also provided a new approach to classify viruses according ot their structure, which could be useful for future cassifications. We envision that these approaches can be used by the scientific community to further classify and characterise these complex viruses.

negative: Coronavirus surveillance of wildlife in the Lao People’s Democratic Republic detects viral RNA in rodents Coronaviruses can become zoonotic as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia facilitates an increased risk for such incidents. We sampled and tested rodents (851) and other mammals, and found Betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters, and relate closely to known rodent coronaviruses detected in the region, and distantly to human coronaviruses OC43 and HKU1. Considering close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks.

negative: CVTree: a phylogenetic tree reconstruction tool based on whole genomes. Composition Vector Tree (CVTree) implements a systematic method of inferring evolutionary relatedness of microbial organisms from the oligopeptide content of their complete proteomes (http://cvtree.cbi.pku.edu.cn). Since the first bacterial genomes were sequenced in 1995 there have been several attempts to infer prokaryote phylogeny from complete genomes. Most of them depend on sequence alignment directly or indirectly and, in some cases, need fine-tuning and adjustment. The composition vector method circumvents the ambiguity of choosing the genes for phylogenetic reconstruction and avoids the necessity of aligning sequences of essentially different length and gene content. This new method does not contain 'free' parameter and 'fine-tuning'. A bootstrap test for a phylogenetic tree of 139 organisms has shown the stability of the branchings, which support the small subunit ribosomal RNA (SSU rRNA) tree of life in its overall structure and in many details. It may provide a quick reference in prokaryote phylogenetics whenever the proteome of an organism is available, a situation that will become commonplace in the near future.

negative: Ultra-sensitive and high-throughput CRISPR-Powered COVID-19 diagnosis Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to amplify target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ∼50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved qPCR assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

negative: Nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies – a comprehensive review Nipah (Nee-pa) viral disease is a zoonotic infection caused by Nipah virus (NiV), a paramyxovirus belonging to the genus Henipavirus of the family Paramyxoviridae. It is a biosafety level-4 pathogen, which is transmitted by specific types of fruit bats, mainly Pteropus spp. which are natural reservoir host. The disease was reported for the first time from the Kampung Sungai Nipah village of Malaysia in 1998. Human-to-human transmission also occurs. Outbreaks have been reported also from other countries in South and Southeast Asia. Phylogenetic analysis affirmed the circulation of two major clades of NiV as based on currently available complete N and G gene sequences. NiV isolates from Malaysia and Cambodia clustered together in NiV-MY clade, whereas isolates from Bangladesh and India clusterered within NiV-BD clade. NiV isolates from Thailand harboured mixed population of sequences. In humans, the virus is responsible for causing rapidly progressing severe illness which might be characterized by severe respiratory illness and/or deadly encephalitis. In pigs below six months of age, respiratory illness along with nervous symptoms may develop. Different types of enzyme-linked immunosorbent assays along with molecular methods based on polymerase chain reaction have been developed for diagnostic purposes. Due to the expensive nature of the antibody drugs, identification of broad-spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs). High pathogenicity of NiV in humans, and lack of vaccines or therapeutics to counter this disease have attracted attention of researchers worldwide for developing effective NiV vaccine and treatment regimens.

negative: First genome sequences of buffalo coronavirus from water buffaloes in Bangladesh We report the complete genome sequences of a buffalo coronavirus (BufCoV HKU26) detected from the faecal samples of two domestic water buffaloes (Bubalus bubalis) in Bangladesh. They possessed 98–99% nucleotide identities to bovine coronavirus (BCoV) genomes, supporting BufCoV HKU26 as a member of Betacoronavirus 1. Nevertheless, BufCoV HKU26 possessed distinct accessory proteins between spike and envelope compared to BCoV. Sugar-binding residues in the N-terminal domain of S protein in BCoV are conserved in BufCoV HKU26.

negative: A call for governments to pause Twitter censorship: a cross-sectional study using Twitter data as social-spatial sensors of COVID-19/SARS-CoV-2 research diffusion Objectives: To determine whether Twitter data can be used as social-spatial sensors to show how research on COVID-19/SARS-CoV-2 diffuses through the population to reach the people that are especially affected by the disease. Design: Cross-sectional bibliometric analysis conducted between 23rd March and 14th April 2020. Setting: Three sources of data were used in the analysis: (1) deaths per number of population for COVID-19/SARS-CoV-2 retrieved from Coronavirus Resource Center at John Hopkins University and Worldometer, (2) publications related to COVID-19/SARS-CoV-2 retrieved from WHO COVID-19 database of global publications, and (3) tweets of these publications retrieved from Altmetric.com and Twitter. Main Outcome(s) and Measure(s): To map Twitter activity against number of publications and deaths per number of population worldwide and in the USA states. To determine the relationship between number of tweets as dependent variable and deaths per number of population and number of publications as independent variables. Results: Deaths per one hundred thousand population for countries ranged from 0 to 104, and deaths per one million population for USA states ranged from 2 to 513. Total number of publications used in the analysis was 1761, and total number of tweets used in the analysis was 751,068. Mapping of worldwide data illustrated that high Twitter activity was related to high numbers of COVID-19/SARS-CoV-2 deaths, with tweets inversely weighted with number of publications. Poisson regression models of worldwide data showed a positive correlation between the national deaths per number of population and tweets when holding the country's number of publications constant (coefficient 0.0285, S.E. 0.0003, p<0.001). Conversely, this relationship was negatively correlated in USA states (coefficient -0.0013, S.E. 0.0001, p<0.001). Conclusions: This study shows that Twitter can play a crucial role in the rapid research response during the COVID-19/SARS-CoV-2 global pandemic, especially to spread research with prompt public scrutiny. Governments are urged to pause censorship of social media platforms during these unprecedented times to support the scientific community's fight against COVID-19/SARS-CoV-2.

negative: A Radiation Mitigator as a Potential Treatment for COVID-19

negative: Angiotensin‐converting enzyme 2 in severe acute respiratory syndrome coronavirus and SARS‐CoV‐2: A double‐edged sword? Human angiotensin‐converting enzyme 2 (ACE2) facilitates cellular entry of severe acute respiratory syndrome coronavirus (SARS‐CoV) and SARS‐CoV‐2 as their common receptor. During infection, ACE2‐expressing tissues become direct targets, resulting in serious pathological changes and progressive multiple organ failure or even death in severe cases. However, as an essential component of renin‐angiotensin system (RAS), ACE2 confers protective effects in physiological circumstance, including maintaining cardiovascular homeostasis, fluid, and electrolyte balance. The absence of protective role of ACE2 leads to dysregulated RAS and thus acute changes under multiple pathological scenarios including SARS. This potentially shared mechanism may also be the molecular explanation for pathogenesis driven by SARS‐CoV‐2. We reasonably speculate several potential directions of clinical management including host‐directed therapies aiming to restore dysregulated RAS caused by ACE2 deficiency. Enriched knowledge of ACE2 learned from SARS and COVID‐19 outbreaks can provide, despite their inherent tragedy, informative clues for emerging pandemic preparedness.

negative: [Molecular diagnosis and phylogenetic analysis of the first MERS case in Turkey]. Coronaviruses (CoV) are enveloped, spherical, single-stranded positive-sense RNA viruses causing mainly respiratory and intestinal infections in animals and humans. Until recently five types of human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E, SARS-CoV) have been known, however a novel CoV has been identified in 2012 in Saudi Arabia. This virus, namely MERS-CoV (Middle East Respiratory Syndrome Coronavirus), was classified within Coronaviridae family, Coronavirinae sub-family, Betacoronavirus genus, clade C. It causes acute respiratory infections in humans and transmits via respiratory route and close contact between humans. The aim of this study was to present the first MERS case from Turkey identified by molecular methods and the results of viral sequence analysis. A 42-year-old male Turkish citizen who worked as an employee in Jeddah, Kingdom of Saudi Arabia, admitted to hospital with the complaints of fever and malaise on 25-26 September 2014. Since his symptoms went on and got worse, he returned to Turkey, and hospitalized in a hospital's intensive care unit in Hatay on 6th of October with the symptoms of fever, malaise, sweating, cough and respiratory distress. He transferred to a university hospital on 8th of October and died on 11th October. The tracheal aspirate sample obtained before he died was sent to Virology Unit of Reference Laboratories of the Turkish Public Health Institution. Detection of viral RNA was performed by using a commercial real-time PCR kit (hCoV-EMC Real-Time RT-PCR, Fast Track Diagnostics, Luxembourg) targeting the MERS-CoV E protein (upE), ORF1a and ORF1b gene regions. The reference method Superscript III One Step RT-PCR (Invitrogen, USA) recommended by World Health Organization (WHO) was also applied for confirmation. Both of the methods yielded positive results for MERS-CoV RNA. For the amplification of nucleocapsid (N) and RNA-dependent RNA polymerase (RdRp) genes, hemi-nested PCR (Invitrogen, ABD) was conducted, followed by sequence analysis of 204 nucleotide part of N gene. Phylogenetic tree of N gene was obtained with the use of MEGA6 software. N gene was chosen as it comprised a two aminoacid deletion in the corresponding published sequence from the patient treated in London, United Kingdom. There was no nucleotide or aminoacid change in our isolate, namely ANK/1079/2014 when compared with human Betacoronavirus 2c EMC/2012 reference strain found in Genbank database. The target gene regions selected in our study (UpE, ORF1a, ORF1b, N and RdRp) which were also recommended by WHO, shown to have high specificity and sensitivity for the diagnosis and confirmation of MERS-CoV, and also recommended by WHO. The previous studies indicated that, the viral genomes detected in the earliest cases of humans (clade A) are genetically distinct from the others (clade B) which were isolated from dromedary camels and humans. In our study, according to phylogenetic analysis of partial N gene segment, isolate ANK/1079/2014 has taken place within clade A. In conclusion, MERS-CoV appears to have limited circulation in Arabian Peninsula and Middle-Eastern countries, it should be considered in mind that travel-related cases may export the virus outside these regions leading autochtonous infections in the other parts of the world.

negative: SARS-CoV-2: Camazotz's Curse The world is currently face to face with a pandemic which is spreading rapidly across the globe caused by SARS-CoV-2, a strain of Coronaviruses (CoVs) belonging to subgenus Sarbecovirus of genus Betacoronavirus. World Health Organisation (WHO) on 11 Feb 20 named this disease caused by SARS-CoV-2 as Covid-19. This pandemic is spreading rapidly and more than 20,00,000 cases have occurred globally. The human Coronaviruses discovered in 1960s were considered potentially harmless endemic viruses with seasonal distribution before late 2002. The CoVs are found in a large number of domestic and wild animals and birds. The first pandemic caused by Coronavirus caused by SARS-CoV was recognized in the late 2002 in Guangdong Province and resulted in widespread morbidity and mortality. This was followed by MERS-CoV which began in 2012 in the Arabian peninsula with multiple outbreaks related to it in various parts of the globe. Various studies have suggested how these viruses made their entry from their natural reservoir bats via intermediate host like civets and camels in case of SARS-CoV and MERS-CoV respectively. The intermediate host of the SARS-CoV-2 still needs to be established. The SARS-CoV-2 has 96.2% similarity to the bat Severe Acute Respiratory Syndrome related-Coronavirus (SARSr-CoV RaTG13). SARS-CoV-2 has been found to be more distant in relation to SARS-CoV (79%) and MERS-CoV (50%). At the whole genome sequence level pangolin CoV and SARSr-CoV RaTG13 show 91.02% and 96.2% similarity with SARS-CoV-2 but the S1 subunit of spike protein of pangolin CoV is more closely related to SARS-CoV-2 than SARSr-CoV RaTG13. The genetic analysis of the currently circulating strains of the pandemic have shown 99.98-100% similarity in their genomes implying a recent shift to humans. The animal source of SARS-CoV-2 needs to be identified to implement control measures in the present pandemic. Also, how the virus moves interspecies will help predict and prevent future pandemics.

negative: Regulated multicistronic expression technology for mammalian metabolic engineering Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering.

negative: Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry To determine the genetic and epidemiological relationship of infectious bronchitis virus (IBV) isolates from commercial poultry to attenuated live IBV vaccines we conducted a phylogenetic network analysis on the full-length S1 sequence for Arkansas (Ark), Massachusetts (Mass) and Delmarva/1639 (DMV/1639) type viruses isolated in 2015 from clinical cases by 3 different diagnostic laboratories. Phylogenetic network analysis of Ark isolates showed two predominant groups linked by 2 mutations, consistent with subpopulations found in commercial vaccines for this IBV type. In addition, a number of satellite groups surrounding the two predominant populations were observed for the Ark type virus, which is likely due to mutations associated with the nature of this vaccine to persist in flocks. The phylogenetic network analysis of Mass-type viruses shows two groupings corresponding to different manufacturers vaccine sequences. No satellite groups were observed for Mass-type viruses, which is consistent with no persistence of this vaccine type in the field. At the time of collection, no vaccine was being used for the DMV/1639 type viruses and phylogenetic network analysis showed a dispersed network suggesting no clear change in genetic distribution. Selection pressure analysis showed that the DMV/1639 and Mass-type strains were evolving under negative selection, whereas the Ark type viruses had evolved under positive selection. This data supports the hypothesis that live attenuated vaccine usage does play a role in the genetic profile of similar IB viruses in the field and phylogenetic network analysis can be used to identify vaccine and vaccine origin isolates, which is important for our understanding of the role live vaccines play in the evolutionary trajectory of those viruses.

negative: High-coverage SARS-CoV-2 genome sequences acquired by target capture sequencing In this study, we designed a set of SARS-CoV-2 enrichment probes to increase the capacity for sequence-based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS-CoV-2 enrichment probe set contains 502 120 nt single-stranded DNA biotin-labeled probes designed based on all available SARS-CoV-2 viral sequences and it can be used to enrich for SARS-CoV-2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from cell culture, three nasopharyngeal swab samples (cycle threshold [Ct ] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID-19 (positive control) and four throat swab samples from patients without COVID-19 (negative controls), respectively. Moreover, based on these high-quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication and its phylogenetic relationship with other selected high-quality samples from the Genome Variation Map. Therefore, this universal SARS-CoV-2 enrichment probe system can capture and enrich SARS-CoV-2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles.

negative: Overwhelming Mutations or SNPs of SARS-CoV-2: A Point of Caution • Mutation studies hits cause for the superior infectious rate of SARS-CoV-2. • SARS-CoV-2 is evolving rapidly with number of mutations and SNPs. • Mutations were found in the ACE2 binding region of SARS-CoV-2 spike glycoprotein. • ORF1ab, ORF8 and spike glycoprotein regions of SARS-CoV-2 are highly mutated. • Overwhelming mutations or SNPs of SARS-CoV-2 alerts drug treatment options.

negative: Machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: COVID-19 case study The 2019 novel coronavirus (renamed SARS-CoV-2, and generally referred to as the COVID-19 virus) has spread to 184 countries with over 1.5 million confirmed cases. Such major viral outbreaks demand early elucidation of taxonomic classification and origin of the virus genomic sequence, for strategic planning, containment, and treatment. This paper identifies an intrinsic COVID-19 virus genomic signature and uses it together with a machine learning-based alignment-free approach for an ultra-fast, scalable, and highly accurate classification of whole COVID-19 virus genomes. The proposed method combines supervised machine learning with digital signal processing (MLDSP) for genome analyses, augmented by a decision tree approach to the machine learning component, and a Spearman's rank correlation coefficient analysis for result validation. These tools are used to analyze a large dataset of over 5000 unique viral genomic sequences, totalling 61.8 million bp, including the 29 COVID-19 virus sequences available on January 27, 2020. Our results support a hypothesis of a bat origin and classify the COVID-19 virus as Sarbecovirus, within Betacoronavirus. Our method achieves 100% accurate classification of the COVID-19 virus sequences, and discovers the most relevant relationships among over 5000 viral genomes within a few minutes, ab initio, using raw DNA sequence data alone, and without any specialized biological knowledge, training, gene or genome annotations. This suggests that, for novel viral and pathogen genome sequences, this alignment-free whole-genome machine-learning approach can provide a reliable real-time option for taxonomic classification.

negative: Genetic diversity and phylogenetic analysis of Feline Coronavirus sequences from Portugal Abstract The distribution of FCoV Types I and II in a Portuguese cat population was studied by a RT-PCR assay targeting the 3′-end of the viral RNA. For a period of 3 years, 120 samples were collected and 57 were found positive for FCoV RNA. Within the positive samples the presence of FCoV Type I was found in 79%. Type II was only detected in 3.5% in animals with Feline Infectious Peritonitis. The remaining 17.5% could not be differentiated. These viral sequences, comprising a region within gene S were further subjected to a heteroduplex mobility assay (HMA) detecting the presence of viral quasispecies in 17% of the samples. Phylogenetic analysis for FCoV Type I revealed high genetic diversity between the Portuguese sequences and other previously characterized strains, while Type II tree showed a higher genetic homogeneity. This study confirmed the presence of FCoV Types I and II circulating in Portugal and detected high genetic diversity between circulating strains suggesting that the virus persists within the host as mixed viral populations.

negative: A Chinese Case of Coronavirus Disease 2019 (COVID-19) Did Not Show Infectivity During the Incubation Period: Based on an Epidemiological Survey Controversy remains over whether the coronavirus disease 2019 (COVID-19) virus may have infectivity during the incubation period before the onset of symptoms. The author had the opportunity to examine the infectivity of COVID-19 during the incubation period by conducting an epidemiological survey on a confirmed patient who had visited Jeju Island during the incubation period. The epidemiological findings support the claim that the COVID-19 virus does not have infectivity during the incubation period.

negative: SARS-CoV-2 is well adapted for humans. What does this mean for re-emergence? In a side-by-side comparison of evolutionary dynamics between the 2019/2020 SARS-CoV-2 and the 2003 SARS-CoV, we were surprised to find that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had developed several advantageous adaptations for human transmission. Our observations suggest that by the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV. However, no precursors or branches of evolution stemming from a less human-adapted SARS-CoV-2-like virus have been detected. The sudden appearance of a highly infectious SARS-CoV-2 presents a major cause for concern that should motivate stronger international efforts to identify the source and prevent near future re-emergence. Any existing pools of SARS-CoV-2 progenitors would be particularly dangerous if similarly well adapted for human transmission. To look for clues regarding intermediate hosts, we analyze recent key findings relating to how SARS-CoV-2 could have evolved and adapted for human transmission, and examine the environmental samples from the Wuhan Huanan seafood market. Importantly, the market samples are genetically identical to human SARS-CoV-2 isolates and were therefore most likely from human sources. We conclude by describing and advocating for measured and effective approaches implemented in the 2002-2004 SARS outbreaks to identify lingering population(s) of progenitor virus.

negative: Detection of Group 1 Coronaviruses in Bats in North America The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People’s Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription–PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

negative: Identification of New Respiratory Viruses in the New Millennium The rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. The inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. In 2001, this led to the discovery of human metapneumovirus (hMPV) and soon following that the outbreak of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (HCoV) NL63 and HCoV-HKU1. Human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. Middle East Respiratory Syndrome coronavirus (MERS-CoV) represents the most recent outbreak of a completely novel respiratory virus, which occurred in Saudi Arabia in 2012 and presents a significant threat to human health. This review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001.

negative: Cloning, expression and characterization of ferret CXCL10 Chemokines and their receptors function in the recruitment and activation of cells of the immune system to sites of inflammation. As such, chemokines play an important role in mediating pathophysiological events during microbial infection. In particular, CXCL9, 10 and 11 and their cognate receptor CXCR3 have been associated with the clinical course of several infectious diseases, including severe acute respiratory syndrome (SARS) and influenza. While CXCL9, 10 and 11 share the same receptor and have overlapping functions, each can also have unique activity in host defense. The lack of a preferred characterized animal model for SARS has brought our attention to ferrets, which have been used for years in influenza studies. The lack of immunological reagents for ferrets prompted us to clone CXCL9, 10, 11 and CXCR3 and, in the case of CXCL10, to express the gene as a recombinant protein. In this study we demonstrate that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS coronavirus. This study therefore represents an important step towards development of the ferret as a model for the role of CXCL9, 10 and 11:CXCR3 axis in severe viral infections.

negative: Ecology, evolution and classification of bat coronaviruses in the aftermath of SARS Abstract In 2002/2003, a novel coronavirus (CoV) caused a pandemic, infecting more than 8000 people, of whom nearly 10% died. This virus, termed severe acute respiratory syndrome-CoV was linked to a zoonotic origin from rhinolophid bats in 2005. Since then, numerous studies have described novel bat CoVs, including close relatives of the newly emerging Middle East respiratory syndrome (MERS)-CoV. In this paper we discuss CoV genomic properties and compare different taxonomic approaches in light of the technical difficulties of obtaining full genomic sequences directly from bat specimens. We first present an overview of the available studies on bat CoVs, with details on their chiropteran hosts, then comparatively analyze the increase in bat CoV studies and novel genomic sequences obtained since the SARS pandemic. We then conduct a comprehensive phylogenetic analysis of the genera Alpha- and Betacoronavirus, to show that bats harbour more CoV diversity than other mammalian hosts and are widely represented in most, but not all parts of the tree of mammalian CoVs. We next discuss preliminary evidence for phylogenetic co-segregation of CoVs and bat hosts encompassing the Betacoronavirus clades b and d, with an emphasis on the sampling bias that exists among bat species and other mammals, then present examples of CoVs infecting different hosts on the one hand and viruses apparently confined to host genera on the other. We also demonstrate a geographic bias within available studies on bat CoVs, and identify a critical lack of information from biodiversity hotspots in Africa, Asia and Latin America. We then present evidence for a zoonotic origin of four of the six known human CoVs (HCoV), three of which likely involved bats, namely SARS-CoV, MERS-CoV and HCoV-229E; compare the available data on CoV pathogenesis in bats to that in other mammalian hosts; and discuss hypotheses on the putative insect origins of CoV ancestors. Finally, we suggest caution with conclusions on the zoonotic potential of bat viruses, based only on genomic sequence data, and emphasize the need to preserve these ecologically highly relevant animals. This paper forms part of a symposium in Antiviral Research on “from SARS to MERS: 10years of research on highly pathogenic human coronaviruses”.

negative: SARS-CoV-2 spike protein predicted to form complexes with host receptor protein orthologues from a broad range of mammals SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.

negative: Regaining perspective on SARS-CoV-2 molecular tracing and its implications During the past three months, a new coronavirus (SARS-CoV-2) epidemic has been growing exponentially, affecting over 100 thousand people worldwide, and causing enormous distress to economies and societies of affected countries. A plethora of analyses based on viral sequences has already been published, in scientific journals as well as through non-peer reviewed channels, to investigate SARS-CoV-2 genetic heterogeneity and spatiotemporal dissemination. We examined all full genome sequences currently available to assess the presence of sufficient information for reliable phylogenetic and phylogeographic studies. Our analysis clearly shows severe limitations in the present data, in light of which any finding should be considered, at the very best, preliminary and hypothesis-generating. Hence the need for avoiding stigmatization based on partial information, and for continuing concerted efforts to increase number and quality of the sequences required for robust tracing of the epidemic.

negative: The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus epidemic in China Abstract The 2019-nCoV is officially called SARS-CoV-2 and the disease is named COVID-19. This viral epidemic in China has led to the deaths of over 1800 people, mostly elderly or those with an underlying chronic disease or immunosuppressed state. This is the third serious Coronavirus outbreak in less than 20 years, following SARS in 2002–2003 and MERS in 2012. While human strains of Coronavirus are associated with about 15% of cases of the common cold, the SARS-CoV-2 may present with varying degrees of severity, from flu-like symptoms to death. It is currently believed that this deadly Coronavirus strain originated from wild animals at the Huanan market in Wuhan, a city in Hubei province. Bats, snakes and pangolins have been cited as potential carriers based on the sequence homology of CoV isolated from these animals and the viral nucleic acids of the virus isolated from SARS-CoV-2 infected patients. Extreme quarantine measures, including sealing off large cities, closing borders and confining people to their homes, were instituted in January 2020 to prevent spread of the virus, but by that time much of the damage had been done, as human-human transmission became evident. While these quarantine measures are necessary and have prevented a historical disaster along the lines of the Spanish flu, earlier recognition and earlier implementation of quarantine measures may have been even more effective. Lessons learned from SARS resulted in faster determination of the nucleic acid sequence and a more robust quarantine strategy. However, it is clear that finding an effective antiviral and developing a vaccine are still significant challenges. The costs of the epidemic are not limited to medical aspects, as the virus has led to significant sociological, psychological and economic effects globally. Unfortunately, emergence of SARS-CoV-2 has led to numerous reports of Asians being subjected to racist behavior and hate crimes across the world.

negative: Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) SARS-CoV-2 is a single-stranded RNA virus of ~30 kb genome size which belongs to genus Coronavirus and family Coronaviridae. SARS-CoV-2 has recently emerged and has been declared as a pandemic by the World Health Organization. Genomic characterization of SARS-CoV-2 has shown that it is of zoonotic origin. The structure of SARS-CoV-2 is found to be similar to SARS-CoV with virion size ranging from 70 to 90 nm. Spike, membrane, and envelope surface viral proteins of coronavirus are embedded in host membrane-derived lipid bilayer encapsulating the helical nucleocapsid comprising viral RNA. The genome comprises of 6–11 open reading frames (ORFs) with 5′ and 3′ flanking untranslated regions (UTRs). Sequence variation among SARS-CoV-2 and SARS-CoV revealed no significant difference in ORFs and nsps. The nsps includes two viral cysteine proteases including papain-like protease (nsp3), chymotrypsin-like, 3C-like, or main protease (nsp5), RNA-dependent RNA polymerase (nsp12), helicase (nsp13), and others likely to be involved in the transcription and replication of SARS-CoV-2. The structure of spike glycoprotein structure of SARS-CoV-2 resembles that of the spike protein of SARS-CoV with an root-mean-square deviation (RMSD) of 3.8 Å. Like SARS-CoV, SARS-CoV-2 uses the ACE2 receptor for internalization and TMPRSS2 serine proteases for S protein priming. Histopathological investigation of tissues from SARS-CoV-2 infected patients showed virus-induced cytopathic effect with signs of acute respiratory distress syndrome in lung cells. This chapter discusses about the morphology, genome organization, replication, and pathogenesis of SARS-CoV-2 that may help us understand the disease that may leads to identification of effective antiviral drugs and vaccines.

negative: Herbaspirillum camelliae sp. nov., a novel endophytic bacterium isolated from Camellia sinensis L Bacterial strain WT00CT is an endophytic bacterium that was isolated from the tea plant (Camellia sinensis L.). The phylogenetic analysis of 16S rRNA genes demonstrated that strain WT00CT was a member of the genus Herbaspirillum. This strain is microaerobic, gram-negative and non-pigmented, and its cells are rod shaped, with a polar flagellum. It grew optimally at 34-37 °C, pH 5.0-8.0 and 0-1.5% NaCl (w/v). The G + C content of its genomic DNA was 62.36 mol%. C16:0, iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 were major fatty acids. The strain WT00CT contained six polar lipids, namely DPG (diphosphatidylglycerol), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PC (phosphatidylcholine), GL (glycolipid) and APL (aminophospholipids), and its respiratory quinone was Q8. The strain WT00CT had a genome size of 6.08 Mb with a total ORF of 5,537, in which one gene cluster (36 genes) encoding a type IV secretion system was absent in other members of the Herbaspirillum genus. ANI values of genomic comparison between the strain WT00CT and other Herbaspirillum species were 75-96%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented here, the strain WT00CT represents a novel species in the Herbaspirillum genus, for which the name Herbaspirillum camelliae sp. nov. is proposed. The type strain of H. camelliae sp. nov. is WT00CT (AB 2018017 T and KCTC 62527 T).

negative: High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity A novel coronavirus (SARS-CoV-2) has emerged to a global pandemic and caused significant damages to public health. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides Ang II to control vasodilatation and permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we wonder how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ~3-10 fold when fluorogenic caspase-1 substrate and Bradykinin-analog peptides were used to characterize ACE2 activity. In addition, the enhancement was mediated by ACE2 binding of RBD domain of SARS-CoV-2 spike. These results highlighted the altered activity of ACE2 during SARS-CoV-2 infection and would shed new lights on the pathogenesis of COVID-19 and its complications for better treatments.

negative: A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01) The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (BJ01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).

negative: Coronavirus phylogeny based on a geometric approach Abstract A novel coronavirus has been identified as the cause of the outbreak of severe acute respiratory syndrome (SARS). Previous phylogenetic analyses based on sequence alignments show that SARS-CoVs form a new group distantly related to the other three groups of previously characterized coronaviruses. In this paper, a geometric approach based on the Z-curve representation of the whole genome sequence is proposed to analyze the phylogenetic relationships of coronaviruses. The evolutionary distances are obtained through measuring the differences among the three-dimensional Z-curves. The Z-curve is approximately described by its geometric center and the associated three eigenvectors, which indicate the center position and the trend of the Z-curve, respectively. Although some information is lost due to the approximate description of the Z-curve, the phylogenetic tree constructed based on these parameters is consistent with those of previous analyses. The present method has the merits of simplicity and intuitiveness, but it is still in its premature stage. Because the phylogenetic relationships are inferred from the whole genome, instead of some individual genes, the present method represents a new direction of phylogeny study in the post-genome era.

negative: The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

negative: Transmission Cycle of SARS-CoV and SARS-CoV-2 Severe acute respiratory syndrome (SARS) is a pandemic that has shocked the world twice over the last two decades caused by a highly transmissible and pathogenic coronavirus (CoV). It causes disease in the lower respiratory tract in humans that was first reported in late 2002 in Guangdong province, China, and later on in December 2019 in Wuhan, China. The two viruses designated as SARS-CoV and SARS-CoV-2, respectively, originated probably from the bat and infected humans via carrier animals. The constant recombination and evolution in the CoV genome may have facilitated their cross-species transmission resulting in recurrent emergence as a pandemic. This chapter intends to accumulate recent findings related to CoV transmission and tentative molecular mechanisms governing the process.

negative: CGAP: a new comprehensive platform for the comparative analysis of chloroplast genomes BACKGROUND: Chloroplast is an essential organelle in plants which contains independent genome. Chloroplast genomes have been widely used for plant phylogenetic inference recently. The number of complete chloroplast genomes increases rapidly with the development of various genome sequencing projects. However, no comprehensive platform or tool has been developed for the comparative and phylogenetic analysis of chloroplast genomes. Thus, we constructed a comprehensive platform for the comparative and phylogenetic analysis of complete chloroplast genomes which was named as chloroplast genome analysis platform (CGAP). RESULTS: CGAP is an interactive web-based platform which was designed for the comparative analysis of complete chloroplast genomes. CGAP integrated genome collection, visualization, content comparison, phylogeny analysis and annotation functions together. CGAP implemented four web servers including creating complete and regional genome maps of high quality, comparing genome features, constructing phylogenetic trees using complete genome sequences, and annotating draft chloroplast genomes submitted by users. CONCLUSIONS: Both CGAP and source code are available at http://www.herbbol.org:8000/chloroplast. CGAP will facilitate the collection, visualization, comparison and annotation of complete chloroplast genomes. Users can customize the comparative and phylogenetic analysis using their own unpublished chloroplast genomes.

negative: Companion vaccine Bioinformatic design tool reveals limited functional genomic variability of SARS-Cov-2 Spike Receptor Binding Domain Tracking the genetic variability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for several reasons, such as to identify target sequences to generate robust vaccines and neutralizing monoclonal antibodies; to track viral genetic temporal and geographic evolution; and to mine for viral variants associated with reduced or increased severity. Several online tools and bioinformatic analyses have been released for this purpose; four main viral clades have been described via phylogenetic analyses. Here, we present an open-source bioinformatic protocol focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Results on the whole GISAID sequence dataset at the time of the writing (April–June 2020) confirms no high variability pressure on the viral genome in terms of quality and quantity of mutations. We then focused our analysis on the receptor-binding domain region of the spike protein and only found a few variants associated with particular geographic locations that should be tracked over time. Finally, additional immunogenomic analyses revealed some variation in mutated epitope MHC compatibility and T-cell recognition for most frequent human HLAs.

negative: Perspectives on the development of neutralizing antibodies against SARS-CoV-2 SARS-CoV-2 gains entry to human cells through its spike (S) protein binding to angiotensin-converting enzyme 2 (ACE2). Therefore, the receptor binding domain (RBD) of the S protein is the primary target for neutralizing antibodies. Selection of broad-neutralizing antibodies against SARS-CoV-2 and SARS-CoV is attractive and might be useful for treating not only COVID-19 but also future SARS-related CoV infections. Broad-neutralizing antibodies, such as 47D11, S309, and VHH-72, have been reported to target a conserved region in the RBD of the S1 subunit. The S2 subunit required for viral membrane fusion might be another target. Due to their small size and high stability, single-domain antibodies might have the ability to be administered by an inhaler making them potentially attractive therapeutics for respiratory infections. A cocktail strategy combining two (or more) antibodies that recognize different parts of the viral surface that interact with human cells might be the most effective.

negative: 27 Evolution of SARS Coronavirus and the Relevance of Modern Molecular Epidemiology Severe acute respiratory syndrome (SARS) is the first major zoonotic infectious disease of the twenty-first century. The disease, originally termed “atypical pneumonia,” was first recognized in November 2002 in Guangdong Province, China, which spread rapidly to 26 countries within 5 months and eventually led to 8096 human infection cases and 774 deaths. A previously unrecognized coronavirus, SARS coronavirus (SARS-CoV), was later identified as the causative agent of the SARS. Subsequent investigation indicated that the source of the human infections was most likely originated from infected wildlife animals traded in the live animal markets, with masked palm civets being considered the most prominent and important carrier. Large-scale culling of civets appeared to have prevented further outbreaks in the region. Further epidemiological studies showed that civets are intermediate hosts of SARS-CoV, and bats are the likely natural reservoir of SARS-CoV and a large number of SARS-like coronaviruses (SL-CoVs). Detailed analysis of the massive SARS-CoV genomic sequence data accumulated indicated that SARS-CoV experienced a strong selection pressure during different outbreak phases in humans as well as interspecies transmission from animals to humans. The spike glycoprotein (S) of SARS-CoV plays a key role in virus–host interaction and hence is a key determinant of interspecies transmission. It has been shown that minor changes of amino acid (aa) residues in the S protein could lead to dramatic changes in virus susceptibility in animal and human hosts. This chapter will focus on the genetics and evolution of SARS-CoVs and SL-CoVs in humans, civets, and bats. The events of SARS outbreaks and the accompanying response activities highlight the importance of modern molecular epidemiology in disease investigation and the urgent need to broaden the screening and investigation of unknown viruses in wildlife animals.

negative: Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2 The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

negative: Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

negative: Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis BACKGROUND: Ensuring accurate diagnosis is essential to limit the spread of the SARS-CoV-2 and for the clinical management of COVID-19. Although real-time reverse transcription polymerase chain reaction (RT- qPCR) is the current recommended laboratory method to diagnose SARS-CoV-2 acute infection, several factors such as requirement of special equipment and skilled staff limit the use of these time-consuming molecular techniques. Recently, several easy to perform rapid antigen detection tests were developed and recommended in some countries as the first line of diagnostic. OBJECTIVES: The aim of this study was to evaluate the performances of the Coris COVID-19 Ag Respi-Strip. Study design. We performed a comparison study by testing nasopharyngeal samples with RT-qPCR and antigen rapid test. RESULTS: 148 nasopharyngeal swabs were tested. Amongst the 106 positive RT-qPCR samples, 32 were detected by the rapid antigen test, given an overall sensitivity of 30.2%. All the samples detected positive with the antigen rapid tests were also positive with RT-qPCR. CONCLUSIONS: Highest viral load is associated with better antigen detection rates. Unfortunately, the overall poor sensitivity of the COVID-19 Ag Respi-Strip does not allow using it alone as the frontline testing for COVID-19 diagnosis.

negative: Structural and functional analysis of a potent sarbecovirus neutralizing antibody SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

negative: Discovery of potential multi-target-directed ligands by targeting host-specific SARS-CoV-2 structurally conserved main protease Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the current COVID-19 pandemic. Worldwide this disease has infected over 2.5 million individuals with a mortality rate ranging from 5 to 10%. There are several efforts going on in the drug discovery to control the SARS-CoV-2 viral infection. The main protease (M(Pro)) plays a critical role in viral replication and maturation, thus can serve as the primary drug target. To understand the structural evolution of M(Pro), we have performed phylogenetic and Sequence Similarity Network analysis, that depicted divergence of Coronaviridae M(Pro) in five clusters specific to viral hosts. This clustering was corroborated with the comparison of M(Pro) structures. Furthermore, it has been observed that backbone and binding site conformations are conserved despite variation in some of the residues. These attributes can be exploited to repurpose available viral protease inhibitors against SARS-CoV-2 M(Pro). In agreement with this, we performed screening of ∼7100 molecules including active ingredients present in the Ayurvedic anti-tussive medicines, anti-viral phytochemicals and synthetic anti-virals against SARS-CoV-2 M(Pro) as the primary target. We identified several natural molecules like δ-viniferin, myricitrin, taiwanhomoflavone A, lactucopicrin 15-oxalate, nympholide A, afzelin, biorobin, hesperidin and phyllaemblicin B that strongly binds to SARS-CoV-2 M(Pro). Intrestingly, these molecules also showed strong binding with other potential targets of SARS-CoV-2 infection like viral receptor human angiotensin-converting enzyme 2 (hACE-2) and RNA dependent RNA polymerase (RdRp). We anticipate that our approach for identification of multi-target-directed ligand will provide new avenues for drug discovery against SARS-CoV-2 infection. Communicated by Ramaswamy H. Sarma

negative: Recent progress in understanding 2019 novel coronavirus (SARS-CoV-2) associated with human respiratory disease: detection, mechanisms and treatment ABSTRACT Viral respiratory diseases such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) always pose a severe threat to people. First identified in late December 2019, a novel coronavirus (2019-nCoV; SARS-CoV-2) has affected many provinces in China and multiple countries worldwide. The viral outbreak has aroused panic and a public-health emergency around the world, and the number of infections continues to rise. However, the causes and consequences of the pneumonia remain unknown. To effectively implement epidemic prevention, early identification and diagnosis are critical to disease control. Here we scrutinise a series of available studies by global scientists on the clinical manifestations, detection methods and treatment options for the disease caused by SARS-CoV-2, named coronavirus disease 2019 (COVID-19), and also propose potential strategies for preventing the infection.

negative: COVID-19: viral–host interactome analyzed by network based-approach model to study pathogenesis of SARS-CoV-2 infection BACKGROUND: Epidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. METHODS: We investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV–host interactome was carried out in order to provide a theoretic host–pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein–protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. RESULTS: Although the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. CONCLUSIONS: In this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.

negative: Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S(1)/S(2) subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

negative: Ecological and evolutionary drivers of hemoplasma infection and bacterial genotype sharing in a Neotropical bat community Most emerging pathogens can infect multiple species, underscoring the importance of understanding the ecological and evolutionary factors that allow some hosts to harbor greater infection prevalence and share pathogens with other species. However, our understanding of pathogen jumps is primarily based around viruses, despite bacteria accounting for the greatest proportion of zoonoses. Because bacterial pathogens in bats (Order: Chiroptera) can have conservation and human health consequences, studies that examine the ecological and evolutionary drivers of bacterial prevalence and barriers to pathogen sharing are crucially needed. We here studied hemotropic Mycoplasma spp. (i.e., hemoplasmas) across a species-rich bat community in Belize over two years. Across 469 bats spanning 33 species, half of individuals and two-thirds of species were hemoplasma positive. Infection prevalence was higher for males and for species with larger body mass and colony sizes. Hemoplasmas displayed high genetic diversity (21 novel genotypes) and strong host specificity. Evolutionary patterns supported co-divergence of bats and bacterial genotypes alongside phylogenetically constrained host shifts. Bat species centrality to the network of shared hemoplasma genotypes was phylogenetically clustered and unrelated to prevalence, further suggesting rare—but detectable—bacterial sharing between species. Our study highlights the importance of using fine phylogenetic scales when assessing host specificity and suggests phylogenetic similarity may play a key role in host shifts for not only viruses but also bacteria. Such work more broadly contributes to increasing efforts to understand cross-species transmission and epidemiological consequences of bacterial pathogens.

negative: Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct four amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability, and transmission.

negative: Microstructure, pathophysiology and potential therapeutics of COVID‐19: A comprehensive review There have been over 7 million cases and almost 413,372 deaths globally due to the novel coronavirus (2019‐nCoV) associated disease COVID‐19, as of June 11, 2020. Phylogenetic analysis suggests that there is a common source for these infections. The overall sequence similarities between the spike protein of 2019‐nCoV and that of SARS‐CoV are known to be around 76‐78% and 73‐76% for whole protein and receptor‐binding domain (RBD), respectively. Thus, they have the potential to serve as drug and/ or vaccine candidate. However, the individual response against 2019‐nCoV differs due to genetic variations in the human population. Understanding the variations in Angiotensin‐converting enzyme 2 (ACE2) and human leukocyte antigen (HLA) that may affect the severity of 2019‐nCoV infection could help in identifying individuals at higher risk from the COVID‐19. A number of potential drugs/vaccines as well as antibody/cytokine‐based therapeutics are running in various developmental stages of preclinical/clinical trials against SARS‐CoV, MERS‐CoV and 2019‐nCoV with substantial cross‐reactivity, which may be used against COVID‐19. For diagnosis, reverse transcription polymerase chain reaction (RT‐PCR) is the gold standard test for initial diagnosis of COVID‐19. Kit based on serological tests are also recommended for investigating the spread of COVID‐19 but it is challenging due to antibodies cross‐reactivity. This review comprehensively summarizes the recent reports available regarding the host‐pathogen interaction, morphological and genomic structure of the virus, and the diagnostic techniques as well as available and potential therapeutics against COVID‐19. This article is protected by copyright. All rights reserved.

negative: SARS-CoV-2 genome evolution exposes early human adaptations The set of mutations observed at the outset of the SARS-CoV-2 pandemic may illuminate how the virus will adapt to humans as it continues to spread. Viruses are expected to quickly acquire beneficial mutations upon jumping to a new host species. Advantageous nucleotide substitutions can be identified by their parallel occurrence in multiple independent lineages and are likely to result in changes to protein sequences. Here we show that SARS-CoV-2 is acquiring mutations more slowly than expected for neutral evolution, suggesting purifying selection is the dominant mode of evolution during the initial phase of the pandemic. However, several parallel mutations arose in multiple independent lineages and may provide a fitness advantage over the ancestral genome. We propose plausible reasons for several of the most frequent mutations. The absence of mutations in other genome regions suggests essential components of SARS-CoV-2 that could be the target of drug development. Overall this study provides genomic insights into how SARS-CoV-2 has adapted and will continue to adapt to humans. SUMMARY In this study we sought signals of evolution to identify how the SARS-CoV-2 genome has adapted at the outset of the COVID-19 pandemic. We find that the genome is largely undergoing purifying selection that maintains its ancestral sequence. However, we identified multiple positions on the genome that appear to confer an adaptive advantage based on their repeated evolution in independent lineages. This information indicates how SARS-CoV-2 will evolve as it diversifies in an increasing number of hosts.

negative: Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. The analysis unravel various amino acid mutations in the viral proteins which may provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.

negative: COVID-19 Outbreak: An Overview BACKGROUND: In late December 2019, Chinese health authorities reported an outbreak of pneumonia of unknown origin in Wuhan, Hubei Province. SUMMARY: A few days later, the genome of a novel coronavirus was released (http://virological.org/t/novel-2019-coronavirus-genome/319; Wuhan-Hu-1, GenBank accession No. MN908947) and made publicly available to the scientific community. This novel coronavirus was provisionally named 2019-nCoV, now SARS-CoV-2 according to the Coronavirus Study Group of the International Committee on Taxonomy of Viruses. SARS-CoV-2 belongs to the Coronaviridae family, Betacoronavirus genus, subgenus Sarbecovirus. Since its discovery, the virus has spread globally, causing thousands of deaths and having an enormous impact on our health systems and economies. In this review, we summarize the current knowledge about the epidemiology, phylogenesis, homology modeling, and molecular diagnostics of SARS-CoV-2. KEY MESSAGES: Phylogenetic analysis is essential to understand viral evolution, whereas homology modeling is important for vaccine strategies and therapies. Highly sensitive and specific diagnostic assays are key to case identification, contact tracing, identification of the animal source, and implementation of control measures.

negative: Near-Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Causing a COVID-19 Case in Peru A near-complete genome sequence was obtained for a novel coronavirus (SARS-CoV-2) strain obtained from an oropharyngeal swab from a Peruvian patient with coronavirus syndrome (COVID-19) who had contact with an individual who had returned to Peru from travel to Italy.

negative: Testing the hypothesis of a recombinant origin of the SARS-associated coronavirus The origin of severe acute respiratory syndrome-associated corona-virus (SARS-CoV) is still a matter of speculation, although more than one year has passed since the onset of the SARS outbreak. In this study, we implemented a 3-step strategy to test the intriguing hypothesis that SARS-CoV might have been derived from a recombinant virus. First, we blasted the whole SARS-CoV genome against a virus database to search viruses of interest. Second, we employed 7 recombination detection techniques well documented in successfully detecting recombination events to explore the presence of recombination in SARS-CoV genome. Finally, we conducted phylogenetic analyses to further explore whether recombination has indeed occurred in the course of coronaviruses history predating the emergence of SARS-CoV. Surprisingly, we found that 7 putative recombination regions, located in Replicase 1ab and Spike protein, exist between SARS-CoV and other 6 coronaviruses: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), human coronavirus 229E (HCoV), murine hepatitis virus (MHV), and avian infectious bronchitis virus (IBV). Thus, our analyses substantiate the presence of recombination events in history that led to the SARS-CoV genome. Like the other coronaviruses used in the analysis, SARS-CoV is also a mosaic structure.

negative: CoVDB: a comprehensive database for comparative analysis of coronavirus genes and genomes The recent SARS epidemic has boosted interest in the discovery of novel human and animal coronaviruses. By July 2007, more than 3000 coronavirus sequence records, including 264 complete genomes, are available in GenBank. The number of coronavirus species with complete genomes available has increased from 9 in 2003 to 25 in 2007, of which six, including coronavirus HKU1, bat SARS coronavirus, group 1 bat coronavirus HKU2, groups 2c and 2d coronaviruses, were sequenced by our laboratory. To overcome the problems we encountered in the existing databases during comparative sequence analysis, we built a comprehensive database, CoVDB (http://covdb.microbiology.hku.hk), of annotated coronavirus genes and genomes. CoVDB provides a convenient platform for rapid and accurate batch sequence retrieval, the cornerstone and bottleneck for comparative gene or genome analysis. Sequences can be directly downloaded from the website in FASTA format. CoVDB also provides detailed annotation of all coronavirus sequences using a standardized nomenclature system, and overcomes the problems of duplicated and identical sequences in other databases. For complete genomes, a single representative sequence for each species is available for comparative analysis such as phylogenetic studies. With the annotated sequences in CoVDB, more specific blast search results can be generated for efficient downstream analysis.

negative: Overview of lethal human coronaviruses Coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. Seven coronaviruses that infect humans have been identified: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Among them, SARS-CoV and MERS-CoV caused outbreaks in 2002 and 2012, respectively. SARS-CoV-2 (COVID-19) is the most recently discovered. It has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. Viruses are genetically simple, yet highly diverse. However, the recent outbreaks of SARS-CoV and MERS-CoV, and the ongoing outbreak of SARS-CoV-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. Only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. This paper mainly focuses on SARS-CoV, MERS-CoV, and SARS-CoV-2. Here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment.

negative: Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal A complete genome sequence was obtained for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab specimen of a Nepalese patient with coronavirus disease 2019 (COVID-19), who had returned to Nepal after traveling to Wuhan, China.

negative: Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections The World Health Organization has recently declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, as pandemic. There is currently a lack of knowledge in the antibody response elicited from SARS-CoV-2 infection. One major immunological question is concerning the antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by using plasma from patients infected by SARS-CoV-2 or SARS-CoV, and plasma obtained from infected or immunized mice. Our results show that while cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses is rare, indicating the presence of non-neutralizing antibody response to conserved epitopes in the spike. Whether these non-neutralizing antibody responses will lead to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding the antigenicity differences between SARS-CoV-2 and SARS-CoV, but also has important implications in vaccine

negative: Broad-spectrum coronavirus antiviral drug discovery Introduction: The highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are lethal zoonotic viruses that have emerged into human populations these past 15 years. These coronaviruses are associated with novel respiratory syndromes that spread from person-to-person via close contact, resulting in high morbidity and mortality caused by the progression to Acute Respiratory Distress Syndrome (ARDS). Areas covered: The risks of re-emergence of SARS-CoV from bat reservoir hosts, the persistence of MERS-CoV circulation, and the potential for future emergence of novel coronaviruses indicate antiviral drug discovery will require activity against multiple coronaviruses. In this review, approaches that antagonize viral nonstructural proteins, neutralize structural proteins, or modulate essential host elements of viral infection with varying levels of efficacy in models of highly pathogenic coronavirus disease are discussed. Expert opinion: Treatment of SARS and MERS in outbreak settings has focused on therapeutics with general antiviral activity and good safety profiles rather than efficacy data provided by cellular, rodent, or nonhuman primate models of highly pathogenic coronavirus infection. Based on lessons learned from SARS and MERS outbreaks, lack of drugs capable of pan-coronavirus antiviral activity increases the vulnerability of public health systems to a highly pathogenic coronavirus pandemic.

negative: Complete genome sequence analysis of canine bocavirus 1 identified for the first time in domestic cats In this study, we investigated the presence of canine bocaviruses (CBoVs) in fecal samples from 105 cats with diarrhea and 92 asymptomatic cats in northeast China. One fecal sample, 17CC0312, collected from an asymptomatic cat, was found to be positive for canine bocavirus 1 (CBoV1). The nearly complete genome of this virus was cloned and sequenced. The viral genome was 5,069 nucleotides (nt) in length and combined four open reading frames (ORFs) in the order 5′-NS1-ORF4-NP1-VP1/VP2-3′. The 17CC0312 virus shared more than 90.3% nucleotide sequence identity with CBoV1 reference sequences and was placed within the CBoV1 group in a phylogenetic tree based on complete genome sequences. Further phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed that this feline CBoV1 strain belongs to CBoV1 lineage 3. These data provide the first molecular evidence of the presence of CBoV1 in a domestic cat and suggest that cats might be carriers of CBoV1.

negative: COVID-19 in healthcare workers in three hospitals in the South of the Netherlands, March 2020 Background: Ten days after the first reported case of SARS-CoV-2 infection in the Netherlands, 3.9% of healthcare workers (HCWs) in nine hospitals located in the South of the Netherlands tested positive for SARS-CoV-2 RNA. The extent of nosocomial transmission that contributed to the HCW infections was unknown. Methods: We combined epidemiological data, collected by means of structured interviews of HCWs, with whole genome sequencing (WGS) of SARS-CoV-2 in clinical samples from HCWs and patients in three of nine hospitals that participated in the HCW screening, to perform an in-depth analysis of sources and modes of transmission of SARS -CoV-2 in HCWs and patients. Results: A total of 1,796 out of 12,022 HCWs (15%) of the three participating hospitals were screened, based on clinical symptoms, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete genome sequences of 50 HCWs and 18 patients. Most sequences grouped in 3 clusters, with 2 clusters displaying local circulation within the region. The observed patterns are most consistent with multiple introductions into the hospitals through community acquired infections, and local amplification in the community. Conclusions: Although direct transmission in the hospitals cannot be ruled out, the data does not support widespread nosocomial transmission as source of infection in patients or healthcare workers.

negative: Molecular Architecture of Early Dissemination and Evolution of the SARS-CoV-2 Virus in Metropolitan Houston, Texas We sequenced the genomes of 320 SARS-CoV-2 strains from COVID-19 patients in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. These genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting Houston. Substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into Houston many times independently by individuals who had traveled from different parts of the country and the world. The majority of viruses are apparent progeny of strains derived from Europe and Asia. We found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. We discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. The data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. Significance COVID-19, the disease caused by the SARS-CoV-2 virus, is a global pandemic. To better understand the first phase of virus spread in metropolitan Houston, Texas, we sequenced the genomes of 320 SARS-CoV-2 strains recovered from COVID-19 patients early in the Houston viral arc. We identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. Some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. Our analysis traces the early molecular architecture of SARS-CoV-2 in Houston, and will help us to understand the origin and trajectory of future infection spikes.

negative: Signals of T(h)2 immune response from COVID-19 patients requiring intensive care

negative: Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus Summary A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to the introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to higher titer as pseudotyped virions. In infected individuals G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, although not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus, and support continuing surveillance of Spike mutations to aid in the development of immunological interventions.

negative: Characterization and membrane gene-based phylogenetic analysis of avian infectious bronchitis virus Chinese strain HH06 A Chinese isolate of avian infectious bronchitis virus (IBV) designated HH06 was isolated from the kidney tissues of a chicken flock experiencing an outbreak of nephritis. In vivo pathogenicity of the IBV isolate HH06 was determined by inoculating specific pathogen-free (SPF) chickens. The clinical signs and related gross lesions of HH06 infected chickens were similar with those of the field-infected chickens. SPF embryonated eggs were inoculated with virus suspension for serial passage and their genomic RNA was extracted. RT-PCR technique was utilized to amplify the M gene sequence encoding membrane protein of IBV. Recombinant plasmid named T-vector-M was constructed via inserting the M gene into the TA cloning vector, pMD 18-T. The sequenced M gene and its deduced amino acid (aa) sequences were compared with the published sequences of reference strains. The M gene is of 687 bp in length encoding the M protein of 228 amino acids with a predicted molecular weight of 25.4 kDa. The sequences of the M gene and M protein share 83.9–97.9% and 83.6–96.5% homologous identities, respectively, compared with 29 IBV reference strains derived from different regions or countries, which revealed that there are still significant variations between strains. Furthermore, a phylogenetic tree based on these M DNA sequences was generated, and the tree topology suggests that some Chinese IBV strains may have a common ancestor; however, HH06 is a new local IBV isolate that is responsible for the field outbreak of nephritis.

negative: Selection of key recommendations for quality indicators describing good quality outbreak response BACKGROUND: The performance of recommended control measures is necessary for quick and uniform infectious disease outbreak control. To assess whether these procedures are performed, a valid set of quality indicators (QIs) is required. The goal of this study was to select a set of key recommendations that can be systematically translated into QIs to measure the quality of infectious disease outbreak response from the perspective of disaster emergency responders and infectious disease control professionals. METHODS: Applying the Rand modified Delphi procedure, the following steps were taken to systematically select a set of key recommendations: extraction of recommendations from relevant literature; appraisal of the recommendations in terms of relevance through questionnaires to experts; expert meeting to discuss recommendations; prioritization of recommendations through a second questionnaire; and final expert meeting to approve the selected set. Infectious disease physicians and nurses, policymakers and communication experts participated in the expert group (n = 48). RESULTS: In total, 54 national and international publications were systematically searched for recommendations, yielding over 200 recommendations. The Rand modified Delphi procedure resulted in a set of 65 key recommendations. The key recommendations were categorized into 10 domains describing the whole response pathway from outbreak recognition to aftercare. CONCLUSION: This study provides a set of key recommendations that represents ‘good quality of response to an infectious disease outbreak’. These key recommendations can be systematically translated into QIs. Organizations and professionals involved in outbreak control can use these QIs to monitor the quality of response to infectious disease outbreaks and to assess in which domains improvement is needed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12879-015-0896-x) contains supplementary material, which is available to authorized users.

negative: Viroporins, Examples of the Two-Stage Membrane Protein Folding Model Viroporins are small, α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.

negative: Failure of the cobas(R) SARS-CoV-2 (Roche) E-gene assay is associated with a C-to-T transition at position 26340 of the SARS-CoV-2 genome Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals, while also clearing essential staff to continue work. At the current time a number of RT-PCR tests have been developed to identify SARS-CoV-2, targeting multiple regions in the viral genome. In comparison to other RNA viruses the mutation rate of SARS-CoV-2 is moderate, however given the large number of transmission chains it is prudent to monitor circulating viruses for mutations that might compromise these tests. Here we report the identification of a C-to-T transition at position 26340 of the SARS-CoV-2 genome which is associated with failure of the cobas(R) SARS-CoV-2 E-gene assay. This variant was detected in four health care workers from the same team. Whole genome sequencing of SARS-CoV-2 showed all four to carry genetically identical viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently on three occasions. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of SNPs which might adversely affect the RT-PCRs used in diagnostics. Additionally, it argues that two regions in the SARS-CoV-2 should be targeted in RT-PCRs to avoid false negatives.

negative: Two-way antigenic cross-reactivity between severe acute respiratory syndrome coronavirus (SARS-CoV) and group 1 animal CoVs is mediated through an antigenic site in the N-terminal region of the SARS-CoV nucleoprotein. In 2002, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known CoVs and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine respiratory CoV [PRCV]) was mediated through the N but not the spike protein, whereas weaker cross-reactivity occurred with feline (feline infectious peritonitis virus) and canine CoVs. Using Escherichia coli-expressed recombinant SARS-CoV N protein and fragments, the cross-reactive region was localized between amino acids (aa) 120 to 208. The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted specifically with SARS convalescent-phase sera but not with negative human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted with antisera to animal CoVs, whereas the fragment comprising aa 360 to 412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of HCoV-NL63 with group 1 CoVs was localized to aa 1 to 39 and at least one other antigenic site in the N-protein C terminus, differing from the cross-reactive region identified in SARS-CoV N protein. The observed cross-reactivity is not a consequence of a higher level of amino acid identity between SARS-CoV and porcine CoV nucleoproteins, because sequence comparisons indicated that SARS-CoV N protein has amino acid identity similar to that of infectious bronchitis virus N protein and shares a higher level of identity with bovine CoV N protein within the cross-reactive region. The TGEV and SARS-CoV N proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV-specific antibody assays for screening animal and human sera.

negative: Identification and Characterization of a Novel Alpaca Respiratory Coronavirus Most Closely Related to the Human Coronavirus 229E In 2007, a novel coronavirus associated with an acute respiratory disease in alpacas (Alpaca Coronavirus, ACoV) was isolated. Full-length genomic sequencing of the ACoV demonstrated the genome to be consistent with other Alphacoronaviruses. A putative additional open-reading frame was identified between the nucleocapsid gene and 3'UTR. The ACoV was genetically most similar to the common human coronavirus (HCoV) 229E with 92.2% nucleotide identity over the entire genome. A comparison of spike gene sequences from ACoV and from HCoV-229E isolates recovered over a span of five decades showed the ACoV to be most similar to viruses isolated in the 1960’s to early 1980’s. The true origin of the ACoV is unknown, however a common ancestor between the ACoV and HCoV-229E appears to have existed prior to the 1960’s, suggesting virus transmission, either as a zoonosis or anthroponosis, has occurred between alpacas and humans.

negative: In-silico analysis of SARS-CoV-2 genomes: Insights from SARS encoded non-coding RNAs Recently a novel coronavirus (SARS-CoV-2) emerged from Wuhan, China and has infected more than 571000 people leading to more than 26000 deaths. Since SARS-CoV-2 genome sequences show similarity with those of SARS, we sought to analyze all the available SARS-CoV-2 genomes based on the insights obtained from SARS genome specifically focusing on non-coding RNAs. Here, results are presented from the dual approach i.e identifying host encoded miRNAs that might regulate viral pathogenesis as well as identifying viral encoded miRNAs that might regulate host cell signaling pathways and aid in viral pathogenesis. Analysis utilizing first approach resulted in the identification of 10 host encoded miRNAs that could target the genome of both the viruses (SARS-CoV-2 and SARS reference genome). Interestingly our analysis revealed that there is significantly higher number of host miRNAs that could target SARS-CoV-2 genome as compared to the SARS reference genome. Results from second approach involving SARS-CoV-2 and SARS reference genome identified a set of virus encoded miRNAs which might regulate host signaling pathways. Our analysis further identified a similar “GA” rich motif in SARS-CoV-2 genome that was shown to play a vital role in lung pathogenesis during severe SARS infections. Hence, we successfully identified human and virus encoded miRNAs that might regulate pathogenesis of both these coronaviruses and the fact that more number of host miRNAs could target SARS-CoV-2 genomes possibly reveal as to why this virus follows mild pathogenesis in healthy individuals. We identified non-coding sequences in SARS-CoV-2 genomes that were earlier reported to contribute towards SARS pathology. The study provides insights into the overlapping sequences among these viruses for their effective inhibition as well as identifying new drug targets that could be used for development of new antivirals.

negative: Genomic characterization of a novel SARS-CoV-2 Abstract A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with human to human transmission and extreme human sickness has been as of late announced from the city of Wuhan in China. Our objectives were to mutation analysis between recently reported genomes at various times and locations and to characterize the genomic structure of SARS-CoV-2 using bioinformatics programs. Information on the variation of viruses is of considerable medical and biological impacts on the prevention, diagnosis, and therapy of infectious diseases. To understand the genomic structure and variations of the SARS-CoV-2. The study analyzed 95 SARS-CoV-2 complete genome sequences available in GenBank, National MicrobiologyData Center (NMDC) and NGDC Genome Warehouse from December-2019 until 05 of April-2020. The genomic signature analysis demonstrates that a strong association between the time of sample collection, location of sample and accumulation of genetic diversity. We found 116 mutations, the three most common mutations were 8782C>T in ORF1ab gene, 28144T>C in ORF8 gene and 29095C>T in the N gene. The mutations might affect the severity and spread of the SARS-CoV-2. The finding heavily supports an intense requirement for additional prompt, inclusive investigations that combine genomic detail, epidemiological information and graph records of the clinical features of patients with COVID-19.

negative: Genome Wide Identification of SARS-CoV Susceptibility Loci Using the Collaborative Cross New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses, including an 8.5Mb locus associated with vascular cuffing on chromosome 3 that contained 23 genes and 13 noncoding RNAs. Integrating phenotypic and genetic data narrowed this region to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation. These data establish the Collaborative Cross platform as a powerful genetic resource for uncovering genetic contributions of complex traits in microbial disease severity, inflammation and virus replication in models of outbred populations.

negative: Viral metagenomic analysis of bushpigs (Potamochoerus larvatus) in Uganda identifies novel variants of Porcine parvovirus 4 and Torque teno sus virus 1 and 2 BACKGROUND: As a result of rapidly growing human populations, intensification of livestock production and increasing exploitation of wildlife habitats for animal agriculture, the interface between wildlife, livestock and humans is expanding, with potential impacts on both domestic animal and human health. Wild animals serve as reservoirs for many viruses, which may occasionally result in novel infections of domestic animals and/or the human population. Given this background, we used metagenomics to investigate the presence of viral pathogens in sera collected from bushpigs (Potamochoerus larvatus), a nocturnal species of wild Suid known to move between national parks and farmland, in Uganda. RESULTS: Application of 454 pyrosequencing demonstrated the presence of Torque teno sus virus (TTSuV), porcine parvovirus 4 (PPV4), porcine endogenous retrovirus (PERV), a GB Hepatitis C–like virus, and a Sclerotinia hypovirulence-associated-like virus in sera from the bushpigs. PCR assays for each specific virus combined with Sanger sequencing revealed two TTSuV-1 variants, one TTSuV-2 variant as well as PPV4 in the serum samples and thereby confirming the findings from the 454 sequencing. CONCLUSIONS: Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs.

negative: Tracing New Clinical Manifestations in Patients with COVID-19 in Chile and Its Potential Relationship with the SARS-CoV-2 Divergence PURPOSE OF REVIEW: In this review, we discuss the current implications of the changing genomic epidemiology of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), etiological agent of the Coronavirus Disease 2019 (COVID-19) and its potential relationship with the change of clinical manifestations in patients with confirmed infection. RECENT FINDINGS: Over the course of the current pandemic, the virus has been found more diverse in new countries. Simultaneously, also new clinical manifestations are observed, particularly more prominent gastrointestinal and neurological findings. SUMMARY: SARS-CoV-2/COVID-19 is changing not only its epidemiology, but also its genomic diversity and clinical manifestations, both aspects coupled, needs to be considered in the study of this ongoing pandemic.

negative: The European Classical Swine Fever Virus Database: Blueprint for a Pathogen-Specific Sequence Database with Integrated Sequence Analysis Tools Molecular epidemiology has become an indispensable tool in the diagnosis of diseases and in tracing the infection routes of pathogens. Due to advances in conventional sequencing and the development of high throughput technologies, the field of sequence determination is in the process of being revolutionized. Platforms for sharing sequence information and providing standardized tools for phylogenetic analyses are becoming increasingly important. The database (DB) of the European Union (EU) and World Organisation for Animal Health (OIE) Reference Laboratory for classical swine fever offers one of the world’s largest semi-public virus-specific sequence collections combined with a module for phylogenetic analysis. The classical swine fever (CSF) DB (CSF-DB) became a valuable tool for supporting diagnosis and epidemiological investigations of this highly contagious disease in pigs with high socio-economic impacts worldwide. The DB has been re-designed and now allows for the storage and analysis of traditionally used, well established genomic regions and of larger genomic regions including complete viral genomes. We present an application example for the analysis of highly similar viral sequences obtained in an endemic disease situation and introduce the new geographic “CSF Maps” tool. The concept of this standardized and easy-to-use DB with an integrated genetic typing module is suited to serve as a blueprint for similar platforms for other human or animal viruses.

negative: Tracking the transmission and evolution of MERS-CoV

negative: SARS-CoV-2 sequence typing, evolution and signatures of selection using CoVa, a Python-based command-line utility The current global pandemic COVID-19, caused by SARS-CoV-2, has resulted in millions of infections worldwide in a few months. Global efforts to tackle this situation have produced a tremendous body of genomic data, which can be used for tracing transmission routes, characterization of isolates, and monitoring variants with potential for unusual virulence. Several groups have analyzed these genomes using different approaches. However, as new data become available, the research community needs a pipeline to perform a set of routine analyses, that can quickly incorporate new genome sequences and update the analysis reports. We developed a programmatic tool, CoVa, with this objective. It is a fast, accurate and user-friendly utility to perform a variety of genome analyses on hundreds of SARS-CoV-2 sequences. Using CoVa, we define a modified sequence typing nomenclature and identify sites under positive selection. Further analysis identified some peptides and sites showing geographical patterns of selection. Specifically, we show differences in sequence type distribution between sequences from India and those from the rest of the world. We also show that several sites show signatures of positive selection uniquely in sequences from India. Preliminary evolutionary analysis, using features that will be incorporated into CoVa in the near future, show a mutation rate of 7.4 × 10−4 substitutions/site/year, confirm a temporal signal with a November 2019 origin of SARS-CoV-2, and a heterogeneity in the geographical distribution of Indian samples.

negative: Bioinformatics studies on a function of the SARS-CoV-2 spike glycoprotein as the binding of host sialic acid glycans SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be “hiding” sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.

negative: SARS-CoV-2 orf1b Gene Sequence in the NTNG1 Gene on Human Chromosome 1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.

negative: SARS-Coronavirus ancestor’s foot-prints in South-East Asian bat colonies and the refuge theory Abstract One of the great challenges in the ecology of infectious diseases is to understand what drives the emergence of new pathogens including the relationship between viruses and their hosts. In the case of the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), several studies have shown coronavirus diversity in bats as well as the existence of SARS-CoV infection in apparently healthy bats, suggesting that bats may be a crucial host in the genesis of this disease. To elucidate the biogeographic origin of SARS-CoV and investigate the role that bats played in its emergence, we amplified coronavirus sequences from bat species captured throughout Thailand and assessed the phylogenetic relationships to each other and to other published coronavirus sequences. To this end, RdRp sequence of Coronavirinae was targeted by RT-PCR in non-invasive samples from bats collected in Thailand. Two new coronaviruses were detected in two bat species: one Betacoronavirus in Hipposideros larvatus and one Alphacoronavirus in Hipposideros armiger. Interestingly, these viruses from South-East Asia are related to those previously detected in Africa (Betacoronavirus-b) or in Europe (Alphacoronavirus & Betacoronavirus-b). These findings illuminate the origin and the evolutionary history of the SARS-CoV group found in bats by pushing forward the hypothesis of a Betacoronavirus spill-over from Hipposideridae to Rhinolophidae and then from Rhinolophidae to civets and Human. All reported Betacoronaviruses-b (SARS-CoV group) of Hipposideridae and Rhinolophidae respectively cluster in two groups despite their broad geographic distribution and the sympatry of their hosts, which is in favor of an ancient and genetically independent evolution of Betacoronavirus-b clusters in these families. Moreover, despite its probable pathogenicity, we found that a Betacoronavirus-b can persistently infect a medium-sized hipposiderid bat colony. These findings illustrate the importance of the host phylogeny and the host/pathogen ecological interactions in the description and the understanding of pathogen emergence. The host’s phylogeny, biogeography and behaviour, combined with already described roles of pathogen plasticity and anthropic changes are likely to be co-factors of disease emergence. Elucidating the common ancestor of Hipposideridae and Rhinolophidae is key to understanding the evolutionary history of actual betacoronaviruses and therefore to get an insight of the deep origin of SARS-CoV.

negative: Cryptic transmission of SARS-CoV-2 in Washington State Following its emergence in Wuhan, China, in late November or early December 2019, the SARS-CoV-2 virus has rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared Coronavirus Disease 2019 (COVID-19) a pandemic. Genome sequencing of SARS-CoV-2 strains allows for the reconstruction of transmission history connecting these infections. Here, we analyze 346 SARS-CoV-2 genomes from samples collected between 20 February and 15 March 2020 from infected patients in Washington State, USA. We found that the large majority of SARS-CoV-2 infections sampled during this time frame appeared to have derived from a single introduction event into the state in late January or early February 2020 and subsequent local spread, strongly suggesting cryptic spread of COVID-19 during the months of January and February 2020, before active community surveillance was implemented. We estimate a common ancestor of this outbreak clade as occurring between 18 January and 9 February 2020. From genomic data, we estimate an exponential doubling between 2.4 and 5.1 days. These results highlight the need for large-scale community surveillance for SARS-CoV-2 introductions and spread and the power of pathogen genomics to inform epidemiological understanding.

negative: Coronavirus phylogeny based on base-base correlation. With more and more complete genome sequences having been released, phylogenetic analysis is entering a new era--that of phylogenomics. In this paper, a novel phylogenomic method, named as Base-Base Correlation (BBC), has been proposed to infer phylogenetic relationships from complete genomes, with particular emphasis on coronavirus phylogeny. Following the high-profile publicity of SARS outbreaks, a renewed interest in coronavirus has been promoted and two novel human coronaviruses (NL63 and HKU1) have been identified. Coronavirus phylogenomics based on BBC is well consistent with that of previous studies. BBC, to study genome information structure based on information theory, provides a novel alignment-free phylogenomic methodology in postgenome informatics.

negative: Receptor recognition and cross-species infections of SARS coronavirus Abstract Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10years of research on highly pathogenic human coronaviruses”.

negative: Managing emerging infectious diseases: Is a federal system an impediment to effective laws? In the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries.

negative: The phylogeny of SARS coronavirus Different tree-building methods consistently place the SARS corona-virus (SARS-CoV) as a basal Group 2 coronavirus rather than as an ungrouped species as concluded by others. Detailed comparisons of the SARS-CoV genomic sequence with those of six other coronaviruses failed to find evidence of recombination or genomic rearrangement using computational methods designed for that purpose.

negative: The architecture of SARS-CoV-2 transcriptome SARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3′ tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2. Highlights We provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing. The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs. We discover at least 41 potential RNA modification sites with an AAGAA motif.

negative: Bioinformatics analysis of SARS coronavirus genome polymorphism BACKGROUND: We have compared 38 isolates of the SARS-CoV complete genome. The main goal was twofold: first, to analyze and compare nucleotide sequences and to identify positions of single nucleotide polymorphism (SNP), insertions and deletions, and second, to group them according to sequence similarity, eventually pointing to phylogeny of SARS-CoV isolates. The comparison is based on genome polymorphism such as insertions or deletions and the number and positions of SNPs. RESULTS: The nucleotide structure of all 38 isolates is presented. Based on insertions and deletions and dissimilarity due to SNPs, the dataset of all the isolates has been qualitatively classified into three groups each having their own subgroups. These are the A-group with "regular" isolates (no insertions / deletions except for 5' and 3' ends), the B-group of isolates with "long insertions", and the C-group of isolates with "many individual" insertions and deletions. The isolate with the smallest average number of SNPs, compared to other isolates, has been identified (TWH). The density distribution of SNPs, insertions and deletions for each group or subgroup, as well as cumulatively for all the isolates is also presented, along with the gene map for TWH. Since individual SNPs may have occurred at random, positions corresponding to multiple SNPs (occurring in two or more isolates) are identified and presented. This result revises some previous results of a similar type. Amino acid changes caused by multiple SNPs are also identified (for the annotated sequences, as well as presupposed amino acid changes for non-annotated ones). Exact SNP positions for the isolates in each group or subgroup are presented. Finally, a phylogenetic tree for the SARS-CoV isolates has been produced using the CLUSTALW program, showing high compatibility with former qualitative classification. CONCLUSIONS: The comparative study of SARS-CoV isolates provides essential information for genome polymorphism, indication of strain differences and variants evolution. It may help with the development of effective treatment.

negative: Coronavirus membrane fusion mechanism offers a potential target for antiviral development The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapies against the causative agent, SARS-CoV-2, and also against other pathogenic coronaviruses (CoV) that have emerged in the past or might appear in future. Researchers are therefore focusing on steps in the CoV replication cycle that may be vulnerable to inhibition by broad-spectrum or specific antiviral agents. The conserved nature of the fusion domain and mechanism across the CoV family make it a valuable target to elucidate and develop pan-CoV therapeutics. In this article, we review the role of the CoV spike protein in mediating fusion of the viral and host cell membranes, summarizing the results of research on SARS-CoV, MERS-CoV, and recent peer-reviewed studies of SARS-CoV-2, and suggest that the fusion mechanism be investigated as a potential antiviral target. We also provide a supplemental file containing background information on the biology, epidemiology, and clinical features of all human-infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.

negative: Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism The spread of COVID-19 caused by the SARS-CoV-2 outbreak has been growing since its first identification in December 2019. The publishing of the first SARS-CoV-2 genome made a valuable source of data to study the details about its phylogeny, evolution, and interaction with the host. Protein-protein binding assays have confirmed that Angiotensin-converting enzyme 2 (ACE2) is more likely to be the cell receptor through which the virus invades the host cell. In the present work, we provide an insight into the interaction of the viral spike Receptor Binding Domain (RBD) from different coronavirus isolates with host ACE2 protein. By calculating the binding energy score between RBD and ACE2, we highlighted the putative jump in the affinity from a progenitor form of SARS-CoV-2 to the current virus responsible for COVID-19 outbreak. Our result was consistent with previously reported phylogenetic analysis and corroborates the opinion that the interface segment of the spike protein RBD might be acquired by SARS-CoV-2 via a complex evolutionary process rather than a progressive accumulation of mutations. We also highlighted the relevance of Q493 and P499 amino acid residues of SARS-CoV-2 RBD for binding to human ACE2 and maintaining the stability of the interface. Moreover, we show from the structural analysis that it is unlikely for the interface residues to be the result of genetic engineering. Finally, we studied the impact of eight different variants located at the interaction surface of ACE2, on the complex formation with SARS-CoV-2 RBD. We found that none of them is likely to disrupt the interaction with the viral RBD of SARS-CoV-2.

negative: Cerebrovascular Accident in a Pediatric Patient Presenting With Influenza Abstract Background Acute ischemic stroke (AIS) in pediatric populations accounts for more than half of pediatric strokes and is associated with significant morbidity and mortality. Pediatric AIS can present with nonspecific symptoms or symptoms that mimic alternate pathology. Case Report A 4-month-old female presented to the emergency department for fever, decreased oral intake, and “limp” appearance after antibiotic administration. She was febrile, tachypneic, and hypoxic. Her skin was mottled with 3-s capillary refill, her anterior fontanelle was tense, and she had mute Babinski reflex bilaterally but was moving all extremities. The patient was hyponatremic, thrombocytopenic, and tested positive for influenza A. A computed tomography scan of the brain revealed an acute infarction involving the right frontal, parietal, temporal, and occipital lobes in addition to hyperdensities concerning for thrombosed cortical veins. The patient was transferred for specialty evaluation and was discharged 2 weeks later on levetiracetam. Why Should an Emergency Physician be Aware of This? Pediatric AIS can present with nonspecific symptoms that mimic alternate pathology. A high level of suspicion is needed so as not to miss the diagnosis of pediatric AIS in the emergency department. A thorough neurologic assessment is warranted, and subtle abnormalities should be investigated further.

negative: Coronavirus infections reported by ProMED, February 2000–January 2020 Abstract Introduction Sources describing the global burden of emerging diseases accurately are still limited. We reviewed coronavirus infections reported by ProMED and assessed the reliability of the data retrieved compared to published reports. We evaluated the effectiveness of ProMED as a source of epidemiological data on coronavirus. Methods Using the keyword “coronavirus” in the ProMED search engine, we reviewed all the information from the reports and collected data using a structured form, including year, country, gender, occupation, the number of infected individuals, and the number of fatal cases. Results We identified 109 entries reported between February 29, 2000 and January 22, 2020. A total of 966 cases were reported, with death reported in 188 cases, suggesting an overall case fatality rate (CFR) of 19.5%. Of 70 cases for which the gender was reported, 47 (67.1%) were male. Most of the cases were reported from China, the United Arab Emirates, and Saudi Arabia, with reports from other countries, including imported cases in Europe and North America. Conclusions Internet-based reporting systems such as ProMED are useful to gather information and synthesize knowledge on emerging infections. Although certain areas need to be improved, ProMED provided useful information about coronaviruses especially during outbreaks.

negative: Topological Analysis of SARS CoV-2 Main Protease There is an urgent necessity of effective medication against SARS CoV-2, which is producing the COVID-19 pandemic across the world. Its main protease (Mpro) represents an attractive pharmacological target due to its involvement in essential viral functions. The crystal structure of free Mpro shows a large structural resemblance with the main protease of SARS CoV (nowadays known as SARS CoV-1). Here we report that as average SARS CoV-2 Mpro is 1900% more sensitive than SARS CoV-1 Mpro in transmitting tiny structural changes across the whole protein through long-range interactions. The largest sensitivity of Mpro to structural perturbations is located exactly around the catalytic site Cys-145, and coincides with the binding site of strong inhibitors. These findings, based on a simplified representation of the protein as a residue network, may help in designing potent inhibitors of SARS CoV-2 Mpro. The main protease of the new coronavirus SARS CoV-2 represents one of the most important targets for the antiviral pharmacological actions againsts COVID-19. This enzyme is essential for the virus due to its proteolytic processing of polyproteins. Here we discover that the main protease of SARS CoV-2 is topologically very similar to that of the SARS CoV-1. This is not surprising taking into account that both proteases differ only in 12 amino acids. However, we remarkable found a topological property of SARS CoV-2 that has increased in more than 1900% repect to its SARS CoV-1 analogue. This property reflects the capacity of the new protease of transmitting perturbations across its domains using long-range interactions. Also remarkable is the fact that the amino acids displaying such increased sensitivity to perturbations are around the binding site of the new protease, and close to its catalytic site. We also show that this sensititivy to perturbations is related to the effects of powerful protease inhibitors. In fact, the strongest inhibitors of the SARS CoV-2 main protease are those that produce the least change of this capacity of transmitting perturbations across the protein. We think that these findings may help in the design of new potent anti-SARS CoV-2 inhibitors.

negative: Direct RNA sequencing and early evolution of SARS-CoV-2 Fundamental aspects of SARS-CoV-2 biology remain to be described, having the potential to provide insight to the response effort for this high-priority pathogen. Here we describe the first native RNA sequence of SARS-CoV-2, detailing the coronaviral transcriptome and epitranscriptome, and share these data publicly. A data-driven inference of viral genetic features and evolutionary rate is also made. The rapid sharing of sequence information throughout the SARS-CoV-2 pandemic represents an inflection point for public health and genomic epidemiology, providing early insights into the biology and evolution of this emerging pathogen.

negative: A 25-Year-Old Sample Contributes the Complete Genome Sequence of Avian Coronavirus Vaccine Strain ArkDPI, Reisolated from Commercial Broilers in the United States Here, we report the complete genome sequence of Avian coronavirus strain ArkDPI of the GI-9 lineage, isolated from broiler chickens in North Georgia in 1994. This is the complete genome sequence of this vaccine strain, reisolated from broilers in the United States.

negative: Origin and Possible Genetic Recombination of the Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China: Phylogenetics and Coalescence Analysis The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe acute respiratory tract infection with a high fatality rate in humans. Coronaviruses are capable of infecting multiple species and can evolve rapidly through recombination events. Here, we report the complete genomic sequence analysis of a MERS-CoV strain imported to China from South Korea. The imported virus, provisionally named ChinaGD01, belongs to group 3 in clade B in the whole-genome phylogenetic tree and also has a similar tree topology structure in the open reading frame 1a and -b (ORF1ab) gene segment but clusters with group 5 of clade B in the tree constructed using the S gene. Genetic recombination analysis and lineage-specific single-nucleotide polymorphism (SNP) comparison suggest that the imported virus is a recombinant comprising group 3 and group 5 elements. The time-resolved phylogenetic estimation indicates that the recombination event likely occurred in the second half of 2014. Genetic recombination events between group 3 and group 5 of clade B may have implications for the transmissibility of the virus.

negative: CD8+ T cell cross-reactivity against SARS-CoV-2 conferred by other coronavirus strains and influenza virus While individuals infected with coronavirus disease 2019 (COVID-19) manifested a broad range in susceptibility and severity to the disease, the pre-existing immune memory of related pathogens can influence the disease outcome. Here, we investigated the potential extent of T cell cross-reactivity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be conferred by other coronaviruses and influenza virus, and generated a map of public and private predicted CD8+ T cell epitopes between coronaviruses. Moreover, to assess the potential risk of self-reactivity and/or diminished T cell response for peptides identical or highly similar to the host, we identified predicted epitopes with high sequence similarity with human proteome. Lastly, we compared predicted epitopes from coronaviruses with epitopes from influenza virus deposited in IEDB to support vaccine development against different virus strains. We believe the comprehensive in silico profile of private and public predicted epitopes across coronaviruses and influenza viruses will facilitate design of vaccines capable of protecting against various viral infections.

negative: First report and phylogenetic analysis of porcine deltacoronavirus in Mexico Porcine deltacoronavirus has caused great economic losses in the swine industry worldwide. In this study, we carried out the first detection, sequencing and characterization of this virus in Mexico. We analysed 885 rectal samples by multiplex RT‐PCR to determine coinfections. In addition, the Spike gene was amplified, sequenced and analysed phylogenetically. We found 85 positive samples for porcine deltacoronavirus, representing 9.6% of the total samples, and we determined that the most frequent coinfection was with porcine epidemic diarrhoea virus (54.1%). Four sequences of Mexican isolates were most closely related to those of the United States. The antigenic regions and the glycosylation site of the strains obtained coincide with those previously reported. This relationship is probably related to the commercial exchange of pigs between the US and Mexico and the geographical proximity of these two countries.

negative: Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus Abstract The availability of monoclonal antibodies (mAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection. A molecular characterization of nine monoclonal antibodies raised in immune mice, using highly purified, inactivated SARS-CoV as the inoculating antigen, is presented in this report. These antibodies are specific for numerous viral protein targets, and six of them are able to effectively neutralize SARS-CoV in vitro, including one with a neutralizing titre of 0.075nM. A phylogenetic analysis of the heavy and light chain sequences reveals that the mAbs share considerable homology. The majority of the heavy chains belong to a single Ig germline V-gene family, while considerably more sequence variation is evident in the light chain sequences. These analyses demonstrate that neutralization ability can be correlated with specific murine VH-gene alleles. For instance, one evident trend is high sequence conservation in the VH chains of the neutralizing mAbs, particularly in CDR-1 and CDR-2. The results suggest that optimization of murine mAbs for neutralization of SARS-CoV infection will likely be possible, and will aid in the development of diagnostic tools and passive treatments for SARS-CoV infection.

negative: Phylogeny of SARS-CoV as inferred from complete genome comparison SARS-CoV, as the pathogeny of severe acute respiratory syndrome (SARS), is a mystery that the origin of the virus is still unknown even a few isolates of the virus were completely sequenced. To explore the genesis of SARS-CoV, the FDOD method previously developed by us was applied to comparing complete genomes from 12 SARS-CoV isolates to those from 12 previously identified coronaviruses and an unrooted phylogenetic tree was constructed. Our results show that all SARS-CoV isolates were clustered into a clique and previously identified coronaviruses formed the other clique. Meanwhile, the three groups of coronaviruses depart from each other clearly in our tree that is consistent with the results of prevenient papers. Differently, from the topology of the phylogenetic tree we found that SARS-CoV is more close to group 1 within genus coronavirus. The topology map also shows that the 12 SARS-CoV isolates may be divided into two groups determined by the association with the SARS-CoV from the Hotel M in Hong Kong that may give some information about the infectious relationship of the SARS.

negative: Comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases BACKGROUND: Rodents represent around 43% of all mammalian species, are widely distributed, and are the natural reservoirs of a diverse group of zoonotic viruses, including hantaviruses, Lassa viruses, and tick-borne encephalitis viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. RESULTS: We used next-generation sequencing metagenomic analysis to survey for a range of mammalian viral families in rodents and other small animals of the orders Rodentia, Lagomorpha, and Soricomorpha in China. We sampled 3,055 small animals from 20 provinces and then outlined the spectra of mammalian viruses within these individuals and the basic ecological and genetic characteristics of novel rodent and shrew viruses among the viral spectra. Further analysis revealed that host taxonomy plays a primary role and geographical location plays a secondary role in determining viral diversity. Many viruses were reported for the first time with distinct evolutionary lineages, and viruses related to known human or animal pathogens were identified. Phylogram comparison between viruses and hosts indicated that host shifts commonly happened in many different species during viral evolutionary history. CONCLUSIONS: These results expand our understanding of the viromes of rodents and insectivores in China and suggest that there is high diversity of viruses awaiting discovery in these species in Asia. These findings, combined with our previous bat virome data, greatly increase our knowledge of the viral community in wildlife in a densely populated country in an emerging disease hotspot. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s40168-018-0554-9) contains supplementary material, which is available to authorized users.

negative: Structural modeling of 2019-novel coronavirus (nCoV) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to SARS-CoV and related SARS-like coronaviruses The 2019 novel coronavirus (2019-nCoV) is currently causing a widespread outbreak centered on Hubei province, China and is a major public health concern. Taxonomically 2019-nCoV is closely related to SARS-CoV and SARS-related bat coronaviruses, and it appears to share a common receptor with SARS-CoV (ACE-2). Here, we perform structural modeling of the 2019-nCoV spike glycoprotein. Our data provide support for the similar receptor utilization between 2019-nCoV and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains, which we predict to be proteolytically-sensitive. We suggest this loop confers fusion activation and entry properties more in line with MERS-CoV and other coronaviruses, and that the presence of this structural loop in 2019-nCoV may affect virus stability and transmission.

negative: A Simple Method for Detection of a Novel Coronavirus (SARS-CoV-2) using One-step RT-PCR followed by Restriction Fragment Length Polymorphism BACKGROUND: A novel coronavirus associated with acute respiratory disease (named SARS-CoV-2) is recently identified in Wuhan city, China, spread rapidly worldwide. An early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. OBJECTIVES: We aimed to establish a simple method for detection of SARS-CoV-2 in differentiating with SARS-CoV. STUDY DESIGN: Primers of our in-house RT-PCR assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I and AluI to distinguish between SARS-CoV-2 and SARS-CoV. RESULTS AND DISCUSSIONS: In this report, a 396 bp fragment of the RdRp gene and 345 bp fragment of the E gene were amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP amplified product of SARS-CoV-2 generating 3 fragments of 45, 154 and 197 bp, but it did not cut the amplicon of SARS-CoV. In contrast, the amplified product of SARS-CoV was digested with EcoRI producing 2 fragments of 76 and 320 bp, whereas, the amplicon of SARS-CoV-2 was undigested by Tsp45I help to distinguish clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS-CoV-2 generating 2 fragments of 248 and 97 bp without cutting to SARS-CoV. Accuracy of assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS-CoV-2 in comparison with other reference assays. In conclusion, the present study, we successfully developed a simple method for molecular detection of SARS-CoV-2 in differentiating with SARS-CoV. This article is protected by copyright. All rights reserved.

negative: Comparative transcriptome analysis reveals the intensive early-stage responses of host cells to SARS-CoV-2 infection Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a widespread outbreak of highly pathogenic COVID-19. It is therefore important and timely to characterize interactions between the virus and host cell at the molecular level to understand its disease pathogenesis. To gain insights, we performed high-throughput sequencing that generated time-series data simultaneously for bioinformatics analysis of virus genomes and host transcriptomes implicated in SARS-CoV-2 infection. Our analysis results showed that the rapid growth of the virus was accompanied by an early intensive response of host genes. We also systematically compared the molecular footprints of the host cells in response to SARS-CoV-2, SARS-CoV and MERS-CoV. Upon infection, SARS-CoV-2 induced hundreds of up-regulated host genes hallmarked by a significant cytokine production followed by virus-specific host antiviral responses. While the cytokine and antiviral responses triggered by SARS-CoV and MERS-CoV were only observed during the late stage of infection, the host antiviral responses during the SARS-CoV-2 infection were gradually enhanced lagging behind the production of cytokine. The early rapid host responses were potentially attributed to the high efficiency of SARS-CoV-2 entry into host cells, underscored by evidence of a remarkably up-regulated gene expression of TPRMSS2 soon after infection. Taken together, our findings provide novel molecular insights into the mechanisms underlying the infectivity and pathogenicity of SARS-CoV-2.

negative: Identification and Genetic Characterization of a Novel Respirovirus in Alpine Chamois (Rupicapra rupicapra rupicapra) SIMPLE SUMMARY: The Alpine chamois is a free-living wild ruminant distributed across the mountainous areas of France, Italy, Switzerland, Liechtenstein, Germany, Austria, Slovenia and Croatia. This wide distribution favours interactions with other wild ruminants and livestock, implying the risk of cross-transmission of pathogens. Due to the impact of lung diseases on chamois populations, the investigation of respiratory pathogens is important for wildlife conservation and for the understanding of infection transmission at the livestock–wildlife interface. In the present study, a novel respirovirus was isolated from a chamois with lung lesions and characterized by full-genome sequence and phylogeny. The genome characterization of this novel virus revealed similarities to domestic ruminant respiroviruses, mainly of caprine and ovine origin. Overall, phylogenetic analyses indicated that the chamois virus is distinct from already defined species and suggested that it is a putative novel member of the genus Respirovirus. The present investigation contributes to the knowledge of respiratory infections in wild ruminants and raises questions on the epidemiological link between chamois and other animal species. ABSTRACT: The Respirovirus genus, family Paramamixoviridae, includes respiratory viral pathogens. Here we report the identification and genetic characterization of a respirovirus in an Alpine chamois showing interstitial pneumonia associated with catarrhal bronchopneumonia. The full-genome characterization of this respirovirus, named ChamoisRV/IT2014, revealed low similarities to caprine respirovirus (77.1%), bovine respirovirus (74.5%) and human respirovirus (72.0%). The phylogenetic analyses based on the full-length genome sequence of the novel isolate and reference respirovirus strains showed that ChamoisRV/IT2014 clustered with caprine respirovirus but formed a separate branch. The phylogenetic tree topology of complete large protein amino acid sequences, representing the current species demarcation criterion for Respirovirus genus, showed a 0.05 branch length of ChamoisRV/IT2014 sequence between the nearest node and the tip of the branch, suggesting that this virus belongs to a novel species. This new isolate in a new host species raises several questions to be addressed on the epidemiological role of chamois and the risks of cross-transmission between wild ruminants and livestock.

negative: Cross host transmission in the emergence of MERS coronavirus Coronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact that closely related viruses have been isolated from this but not any other animal species. Given the widespread geographical distribution of dromedaries found seropositive for MERS-CoV, continued transmission may likely occur in the future. Therefore, a further understanding of the cross host transmission of MERS-CoV is needed to limit the risks this virus poses to man.

negative: Adding the spatial dimension to the social network analysis of an epidemic: Investigation of the 2007 outbreak of equine influenza in Australia Abstract Equine influenza is a highly contagious and widespread viral respiratory disease of horses and other equid species, characterised by fever and a harsh dry cough. In 2007, in the first reported outbreak in Australia, the virus spread through the horse populations of two states within 4 months. Most of the geographic spread occurred within the first 10 days and was associated with the movement of infected horses prior to the implementation of movement controls. This study applies social network analysis to describe spread of equine influenza between horse premises infected in the early outbreak period, identifying spread occurring through a contact network and secondary local spatial spread. Social networks were constructed by combining contact-tracing data on horse movements with a distance matrix between all premises holding horses infected within the first 10 days of the outbreak. These networks were analysed to provide a description of the epidemic, identify premises that were central to disease spread and to estimate the relative proportion of premises infected through infected horse movements and through local spatial spread. We then explored the effect of distance on disease spread by estimating the range of local spread (through direct contact, transmission on fomites and windborne transmission) based on the level of fragmentation in the network and also by directly estimating the shape of the outbreak's spatial transmission kernel. During the first 10 days of this epidemic, 197 horse premises were infected; 70 of these were included in the contact-traced network. Most local spread occurred within 5km. Local spread was estimated to have occurred up to a distance of 15.3km – based on the contact-and-proximity network – and at a very low incidence beyond this distance based on the transmission kernel estimate. Of the 70 premises in the contact network, spread to 14 premises (95% CI: 9, 20 premises) was likely to have occurred through local spatial spread from nearby infected premises, suggesting that 28.3% of spread in the early epidemic period was ‘network-associated’ (95% CI: 25.6, 31.0%). By constructing a ‘maximal network’ of contact and proximity (based on a distance cut-off of 15.3km), 44 spatial clusters were described, and the horse movements that initiated infection in these locations were identified. Characteristics of the combined network, incorporating both spatial and underlying contact relationships between infected premises, explained the high rate of spread, the sequence of cluster formation and the widespread dispersal experienced in the early phase of this epidemic. These results can inform outbreak control planning by guiding the imposition of appropriate control zone diameters around infected premises and the targeting of surveillance and interventions.

negative: Temporal evolution and adaptation of SARS-COV 2 codon usage The outbreak of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has caused an unprecedented pandemic. Since the first sequenced whole-genome of SARS-CoV-2 on January 2020, the identification of its genetic variants has become crucial in tracking and evaluating their spread across the globe. In this study, we compared 15,259 SARS-CoV-2 genomes isolated from 60 countries since the outbreak of this novel coronavirus with the first sequenced genome in Wuhan to quantify the evolutionary divergence of SARS-CoV-2. Thus, we compared the codon usage patterns, every two weeks, of 13 of SARS-CoV-2 genes encoding for the membrane protein (M), envelope (E), spike surface glycoprotein (S), nucleoprotein (N), non-structural 3C-like proteinase (3CLpro), ssRNA-binding protein (RBP), 2’-O-ribose methyltransferase (OMT), endoRNase (RNase), helicase, RNA-dependent RNA polymerase (RdRp), Nsp7, Nsp8, and exonuclease ExoN. As a general rule, we find that SARS-CoV-2 genome tends to diverge over time by accumulating mutations on its genome and, specifically, on the coding sequences for proteins N and S. Interestingly, different patterns of codon usage were observed among these genes. Genes S, Nsp7, NSp8, tend to use a norrower set of synonymous codons that are better optimized to the human host. Conversely, genes E and M consistently use a broader set of synonymous codons, which does not vary with respect to the reference genome. We identified key SARS-CoV-2 genes (S, N, ExoN, RNase, RdRp, Nsp7 and Nsp8) suggested to be causally implicated in the virus adaptation to the human host.

negative: COVID-19 diagnostic approaches: different roads to the same destination “SARS-CoV2”, a previously unknown strain of coronaviruses caused a severe respiratory disease called Coronavirus disease (COVID-19) which emerged from Wuhan city of China on 30 December 2019, and declared as Global health problem by World Health Organisation within a month. In less than two and half months (11 March, 2020) it was declared as a pandemic disease due to its rapid spreading ability, it covered more than 211 countries infecting around 1.7 million persons and claiming around 1.1 lakhs lives within merely 100 days of its emergence. Containment of the infection of this virus is the only available measure to control the disease as no vaccine or specific antiviral treatment is available. Confirmed detection of the virus followed by isolation of the infected person at the earliest possible is the only measure to prevent this disease. Although there are number of methods available for detection of virus and to combat this disease in the present pandemic situation, but these available diagnostic methods have their own limitations. The speedy and exponential global spread of this disease strongly urges the fast and economic diagnostics tools. Additional to the available diagnostic methods, there is a sudden surge for development of various of methods and platforms to diagnose the COVID-19. The review summarized the advantage and disadvantage of various diagnostic approaches being used presently for COVID-19, newer detection methods in developmental stage and the feasibility of advanced platforms like newer nano-sensor based on-the-spot detection technologies.

negative: Has India met this enemy before? From an eternal optimist’s perspective: SARS-CoV-2 OBJECTIVE: We are presently going through a historic and unprecedented crisis for humanity with SARS-CoV-2 causing immense damage to life and world economics. It has been 3 months, since we had the first cluster in China and we felt the need to look into certain regional patterns of transmission of the virus with respect to some distinctive living conditions, incidence of malaria, the genomics of different strains, and its impact on severity. MATERIAL AND METHODS: Data for 107 countries was compiled and correlation analysis was done between incidence of malaria and number of SARS-CoV-2 cases. Possibility of genetic similarity between SARS-CoV-2 and reported zoonotic RNA viruses found associated previously with some Plasmodium species was explored by utilizing NCBI database. RESULTS: We found a significant inverse correlation between SARS-CoV-2 disease burden and incidence of Malaria. Our analysis also showed that a 12 base pair region encoding a part of surface glycoprotein of SARS- CoV-2 aligned with the Plasmodium associated zoonotic viral genome. CONCLUSION: Our analysis suggests a significantly lower SARS-CoV-2 disease burden in Malaria endemic regions and an inverse correlation with incidence of Malaria. The possibility of a pre-existing immunological memory for SARS-CoV-2 in Indians is possible and needs to be explored further

negative: Computational View toward the Inhibition of SARS-CoV-2 Spike Glycoprotein and the 3CL Protease Since the outbreak of the 2019 novel coronavirus disease (COVID-19), the medical research community is vigorously seeking a treatment to control the infection and save the lives of severely infected patients. The main potential candidates for the control of viruses are virally targeted agents. In this short letter, we report our calculations on the inhibitors for the SARS-CoV-2 3CL protease and the spike protein for the potential treatment of COVID-19. The results show that the most potent inhibitors of the SARS-CoV-2 3CL protease include saquinavir, tadalafil, rivaroxaban, sildenafil, dasatinib, etc. Ergotamine, amphotericin b, and vancomycin are most promising to block the interaction of the SARS-CoV-2 S-protein with human ACE-2.

negative: No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2

negative: Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19 Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans. Bullet points We generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19. ACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2. ACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities. We unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry.

negative: On the crucial role of multilingual biomedical databases on epidemic events (SARS-CoV-2 analysis)

negative: A Simple Method for Detection of a Novel Coronavirus (SARS‐CoV‐2) using One‐step RT‐PCR followed by Restriction Fragment Length Polymorphism BACKGROUND: A novel coronavirus associated with acute respiratory disease (named SARS‐CoV‐2) is recently identified in Wuhan city, China, spread rapidly worldwide. An early identification of this novel coronavirus by molecular tools is critical for surveillance and control of the epidemic outbreak. OBJECTIVES: We aimed to establish a simple method for detection of SARS‐CoV‐2 in differentiating with SARS‐CoV. STUDY DESIGN: Primers of our in‐house RT‐PCR assays were designed to target conserved regions of the RdRP gene and E gene, selected restriction enzymes EcoRI, Tsp45I and AluI to distinguish between SARS‐CoV‐2 and SARS‐CoV. RESULTS AND DISCUSSIONS: In this report, a 396 bp fragment of the RdRp gene and 345 bp fragment of the E gene were amplified by one‐step RT‐PCR. Enzyme Tsp45I cuts the RdRP amplified product of SARS‐CoV‐2 generating 3 fragments of 45, 154 and 197 bp, but it did not cut the amplicon of SARS‐CoV. In contrast, the amplified product of SARS‐CoV was digested with EcoRI producing 2 fragments of 76 and 320 bp, whereas, the amplicon of SARS‐CoV‐2 was undigested by Tsp45I help to distinguish clearly SARS‐CoV‐2 from SARS‐CoV on gel electrophoresis. In addition, AluI cut the amplicon of the E gene of SARS‐CoV‐2 generating 2 fragments of 248 and 97 bp without cutting to SARS‐CoV. Accuracy of assay was confirmed by sequencing and phylogenetic analysis. When evaluated on clinical samples showed a high sensitivity of 95%, specificity of our assay was 100% and clinical performance for detection of SARS‐CoV‐2 in comparison with other reference assays. In conclusion, the present study, we successfully developed a simple method for molecular detection of SARS‐CoV‐2 in differentiating with SARS‐CoV. This article is protected by copyright. All rights reserved.

negative: Analysis of SARS-CoV Receptor Activity of ACE2 Orthologs

negative: SARS-CoV originated from bats in 1998 and may still exist in humans SARS-CoV is believed to originate from civets and was thought to have been eliminated as a threat after the 2003 outbreak. Here, we show that human SARS-CoV (huSARS-CoV) originated directly from bats, rather than civets, by a cross-species jump in 1991, and formed a human-adapted strain in 1998. Since then huSARS-CoV has evolved further into highly virulent strains with genotype T and a 29-nt deletion mutation, and weakly virulent strains with genotype C but without the 29-nt deletion. The former can cause pneumonia in humans and could be the major causative pathogen of the SARS outbreak, whereas the latter might not cause pneumonia in humans, but evolved the ability to co-utilize civet ACE2 as an entry receptor, leading to interspecies transmission between humans and civets. Three crucial time points - 1991, for the cross-species jump from bats to humans; 1998, for the formation of the human-adapted SARS-CoV; and 2003, when there was an outbreak of SARS in humans - were found to associate with anomalously low annual precipitation and high temperatures in Guangdong. Anti-SARS-CoV sero-positivity was detected in 20% of all the samples tested from Guangzhou children who were born after 2005, suggesting that weakly virulent huSARS-CoVs might still exist in humans. These existing but undetected SARS-CoVs have a large potential to evolve into highly virulent strains when favorable climate conditions occur, highlighting a potential risk for the reemergence of SARS.

negative: Fractal signature of coronaviruses related to severe acute respiratory syndrome PURPOSE: The aim of this work was to investigate the genome of SARS-CoV, MERS-CoV, and SARS-CoV-2 by the paradigm of chaos theory and fractal geometry. Coronavirus is the agent that causes the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) and emergent infections such as the present pandemic of COVID-19. Understanding its genome pattern is important for developing new and faster ways of testing for identifying the genome of the virus and also for better understanding of its origin and evolution. METHODS: For that, it was calculated the alpha coefficient by detrended fluctuation analysis (DFA) for the sequences of these genomes converted to binary numbers in order to determine if it is a chaotic or a random series of data. Also, it applied the random walking for obtaining a fractal map of the whole genome and calculated the fractal dimension (FD) by box-counting of this map by two different software. RESULTS: With this, it was found that the alpha coefficient of the first SARS viruses was > 0.5, indicating that the series is chaotic or fractal, and has a persistent long-range memory or self-similarity along its sequence. This is not the case for MERS virus, which showed to have a completely random sequence (α < 0.5). For the fractal dimension, SARS viruses presented a FD around 1.5, and for MERS the fractal dimension decreases (FD < 1.5). CONCLUSION: The images generated by random walking of the entire RNA genome are by itself a fractal signature of the virus, which may be applied for studying its origin and for faster diagnostic of COVID-19.

negative: Complete genome sequence analysis of a predominant infectious bronchitis virus (IBV) strain in China Infectious bronchitis (IB) is one of the major diseases in poultry flocks all over the world caused by infectious bronchitis virus (IBV). In the study, the complete genome sequence of strain A2 was sequenced and analyzed, which was a predominant IBV strain in China. The results indicated that there were mutations, insertions, and deletions distributed in the whole genome. The A2 virus had the highest identity to S14 and BJ in terms of full genome, whereas had a further distance to Massachusetts strains. Phylogenetic analysis showed that A2 isolate clustered together with most Chinese strains. The results of this study suggest that strain A2 may play an important role in IBV’s evolution and A2-like IBVs are predominant strains in China.

negative: An emergent clade of SARS-CoV-2 linked to returned travellers from Iran The SARS-CoV-2 epidemic has rapidly spread outside China with major outbreaks occurring in Italy, South Korea, and Iran. Phylogenetic analyses of whole-genome sequencing data identified a distinct SARS-CoV-2 clade linked to travellers returning from Iran to Australia and New Zealand. This study highlights potential viral diversity driving the epidemic in Iran, and underscores the power of rapid genome sequencing and public data sharing to improve the detection and management of emerging infectious diseases.

negative: Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2 Abstract SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.

negative: Shared Common Ancestry of Rodent Alphacoronaviruses Sampled Globally The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features, such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species.

negative: A High-Coverage SARS-CoV-2 Genome Sequence Acquired by Target Capture Sequencing This manuscript is based on the method we developed urgently to deal with the research requirement in the conflict between achieving a complete genome sequence for the evolutionary history of SARS-CoV-2 study and the low viral RNA concentration. Here, in this manuscript, we developed a set of SARS-CoV-2 enrichment probes to increase the sensitivity of sequence-based virus detection and characterization via obtaining the comprehensive genome sequence. Following the CDC health and safety guidelines, we test the concept using the culturing supernatant contain SARS-CoV-2 particles, and its full-length sequence was used for further analysis. The fraction of SARS-CoV-2 endogenous DNA was 93.47% with Cluster Factor about 1.1, which demonstrate that the numbers of mapped reads to SARS-CoV-2 reference sequence significantly increased, compared to metagenomic sequencing technology, following SARS-CoV-2 probe enrichment. Moreover, based on the high-quality sequence, we discussed the heterozygosity and viral expression during replication of coronavirus, and its phylogenetic relationship with other selected high-quality samples from The Genome Variation Map (GVM) (on 2020/03/22). We believe this manuscript is valuable for all the researchers who are interested in using clinical warp samples to obtain the high coverage of SARS-CoV-2 genome sequence with a relatively low concentration of viral particles. This would allow the clinician to correlate the diagnostic data with molecular monitoring in viral evolutional, the most importantly, to track the functional mutation of SARS-CoV-2.

negative: Human monoclonal IgG selection of Plasmodium falciparum for the expression of placental malaria-specific variant surface antigens Pregnancy-associatedPlasmodium falciparum malaria (PAM) is a major cause of morbidity and mortality in African women and their offspring. PAM is characterized by accumulation of infected erythrocytes (IEs) that adhere to chondroitin sulphate A (CSA) in the placental intervillous space. We show here that human monoclonal IgG antibodies with specificity for variant surface antigens (VSA) specifically expressed by CSA-adhering IEs (VSA(PAM)) can be used in vitro to select parasites from nonpregnant donors to express VSA(PAM) and that this selection for VSA(PAM) expression results in preferential transcription of var2csa. The results corroborate current efforts to develop PAM-specific vaccines based on VAR2CSA.

negative: A distinct name is needed for the new coronavirus

negative: What was the primary mode of smallpox transmission? Implications for biodefense The mode of infection transmission has profound implications for effective containment by public health interventions. The mode of smallpox transmission was never conclusively established. Although, “respiratory droplet” transmission was generally regarded as the primary mode of transmission, the relative importance of large ballistic droplets and fine particle aerosols that remain suspended in air for more than a few seconds was never resolved. This review examines evidence from the history of variolation, data on mucosal infection collected in the last decades of smallpox transmission, aerosol measurements, animal models, reports of smallpox lung among healthcare workers, and the epidemiology of smallpox regarding the potential importance of fine particle aerosol mediated transmission. I introduce briefly the term anisotropic infection to describe the behavior of Variola major in which route of infection appears to have altered the severity of disease.

negative: SARS-CoV-2 virus RNA sequence classification and geographical analysis with convolutional neural networks approach Covid-19 infection, which spread to the whole world in December 2019 and is still active, caused more than 250 thousand deaths in the world today. Researches on this subject have been focused on analyzing the genetic structure of the virus, developing vaccines, the course of the disease, and its source. In this study, RNA sequences belonging to the SARS-CoV-2 virus are transformed into gene motifs with two basic image processing algorithms and classified with the convolutional neural network (CNN) models. The CNN models achieved an average of 98% Area Under Curve(AUC) value was achieved in RNA sequences classified as Asia, Europe, America, and Oceania. The resulting artificial neural network model was used for phylogenetic analysis of the variant of the virus isolated in Turkey. The classification results reached were compared with gene alignment values in the GISAID database, where SARS-CoV-2 virus records are kept all over the world. Our experimental results have revealed that now the detection of the geographic distribution of the virus with the CNN models might serve as an efficient method.

negative: Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak An outbreak of coronavirus disease 2019 (COVID-19) caused by the 2019 novel coronavirus (SARS-CoV-2) began in the city of Wuhan in China and has widely spread worldwide. Currently, it is vital to explore potential intermediate hosts of SARS-CoV-2 to control COVID-19 spread. Therefore, we reinvestigated published data from pangolin lung samples from which SARS-CoV-like CoVs were detected by Liu et al. [1]. We found genomic and evolutionary evidence of the occurrence of a SARS-CoV-2-like CoV (named Pangolin-CoV) in dead Malayan pangolins. Pangolin-CoV is 91.02% and 90.55% identical to SARS-CoV-2 and BatCoV RaTG13, respectively, at the whole-genome level. Aside from RaTG13, Pangolin-CoV is the most closely related CoV to SARS-CoV-2. The S1 protein of Pangolin-CoV is much more closely related to SARS-CoV-2 than to RaTG13. Five key amino acid residues involved in the interaction with human ACE2 are completely consistent between Pangolin-CoV and SARS-CoV-2, but four amino acid mutations are present in RaTG13. Both Pangolin-CoV and RaTG13 lost the putative furin recognition sequence motif at S1/S2 cleavage site that can be observed in the SARS-CoV-2. Conclusively, this study suggests that pangolin species are a natural reservoir of SARS-CoV-2-like CoVs.

negative: Diversity of coronavirus in bats from Eastern Thailand BACKGROUND: Bats are reservoirs for a diverse range of coronaviruses (CoVs), including those closely related to human pathogens such as Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome CoV. There are approximately 139 bat species reported to date in Thailand, of which two are endemic species. Due to the zoonotic potential of CoVs, standardized surveillance efforts to characterize viral diversity in wildlife are imperative. FINDINGS: A total of 626 bats from 19 different bat species were individually sampled from 5 provinces in Eastern Thailand between 2008 and 2013 (84 fecal and 542 rectal swabs). Samples collected (either fresh feces or rectal swabs) were placed directly into RNA stabilization reagent, transported on ice within 24 hours and preserved at −80°C until further analysis. CoV RNA was detected in 47 specimens (7.6%), from 13 different bat species, using broadly reactive consensus PCR primers targeting the RNA-Dependent RNA Polymerase gene designed to detect all CoVs. Thirty seven alphacoronaviruses, nine lineage D betacoronaviruses, and one lineage B betacoronavirus (SARS-CoV related) were identified. Six new bat CoV reservoirs were identified in our study, namely Cynopterus sphinx, Taphozous melanopogon, Hipposideros lekaguli, Rhinolophus shameli, Scotophilus heathii and Megaderma lyra. CONCLUSIONS: CoVs from the same genetic lineage were found in different bat species roosting in similar or different locations. These data suggest that bat CoV lineages are not strictly concordant with their hosts. Our phylogenetic data indicates high diversity and a complex ecology of CoVs in bats sampled from specific areas in eastern regions of Thailand. Further characterization of additional CoV genes may be useful to better describe the CoV divergence.

negative: Molecular evolution and multilocus sequence typing of 145 strains of SARS-CoV Abstract In this study, we have identified 876 polymorphism sites in 145 complete or partial genomes of SARS-CoV available in the NCBI GenBank. One hundred and seventy-four of these sites existed in two or more SARS-CoV genome sequences. According to the sequence polymorphism, all SARS-CoVs can be divided into three groups: (I) group 1, animal-origin viruses (such as SARS-CoV SZ1, SZ3, SZ13 and SZ16); (II) group 2, all viruses with clinical origin during first epidemic; and (III) group 3, SARS-CoV GD03T0013. According to 10 special loci, group 2 again can be divided into genotypes C and T, which can be further divided into sub-genotypes C1–C4 and T1–T4. Positive Darwinian selections were identified between any pair of these three groups. Genotype C gives neutral selection. Genotype T, however, shows negative selection. By comparing the death rates of SARS patients in the different regions, it was found that the death rate caused by the viruses of the genotype C was lower than that of the genotype T. SARS-CoVs might originate from an unknown ancestor.

negative: The Architecture of SARS-CoV-2 Transcriptome Summary SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3′ tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

negative: MicroGMT: A Mutation Tracker for SARS-CoV-2 and Other Microbial Genome Sequences With the continued spread of SARS-CoV-2 virus around the world, researchers often need to quickly identify novel mutations in newly sequenced SARS-CoV-2 genomes for studying the molecular evolution and epidemiology of the virus. We have developed a Python package, MicroGMT, which takes either raw sequence reads or assembled genome sequences as input and compares against database sequences to identify and characterize indels and point mutations. Although our default setting is optimized for SARS-CoV-2 virus, the package can be also applied to any other microbial genomes. The software is freely available at Github URL https://github.com/qunfengdong/MicroGMT.

negative: Molecular characterization of Brazilian wild-type strains of bovine respiratory syncytial virus reveals genetic diversity and a putative new subgroup of the virus Background: Bovine orthopneumovirus, formerly known as bovine respiratory syncytial virus (BRSV), is frequently associated with bovine respiratory disease (BRD). Aim: To perform the molecular characterization of the G and F proteins of Brazilian wild-type BRSV strains derived from bovine respiratory infections in both beef and dairy cattle. Materials and Methods: Ten BRSV strains derived from a dairy heifer rearing unit (n = 3) in 2011 and steers of three other feedlots (n = 7) in 2014 and 2015 were analyzed. For the BRSV G and F partial gene amplifications, RT-nested-PCR assays were performed with sequencing in both directions with forward and reverse primers used. Results: The G gene-based analysis revealed that two strains were highly similar to the BRSV sequences representative of subgroup III, including the Bayovac vaccine strain. However, the remaining seven Brazilian BRSV strains were diverse when compared with strains representative of the BRSV I to VIII subgroups. The central hydrophobic region of the Brazilian BRSV G gene showed the replacement of conserved cysteines and other residues of importance to antibody reactivity. The deduced F gene amino acid sequences from the Brazilian BRSV strains showed changes that were absent in the representative sequences of the known subgroups. Viral isolation on the nasopharyngeal swab suspensions failed to isolate BRSV. Conclusion: Results suggest that these strains represent a putative new subgroup of BRSV with mutations observed in the immunodominant region of the G protein. However, further studies on these Brazilian BRSV strains should be performed to establish their pathogenic potential.

negative: Broad neutralization of SARS-related viruses by human monoclonal antibodies Broadly protective vaccines against known and pre-emergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

negative: Molecular analysis of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43 Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

negative: Evolutionary Relationships between Bat Coronaviruses and Their Hosts Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (CoVs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (SARS) CoV, which caused an outbreak of respiratory illness in humans during 2002–2003. We examined the evolutionary relationships between bat CoVs and their hosts by using sequence data of the virus RNA-dependent RNA polymerase gene and the bat cytochrome b gene. Phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their CoVs, which suggested that host shifts have occurred in the recent evolutionary history of this group. These shifts may be due to either virus biologic traits or host behavioral traits. This finding has implications for the emergence of SARS and for the potential future emergence of SARS-CoVs or related viruses.

negative: A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease The duration and nature of immunity generated in response to SARS-CoV-2 infection is unknown. Many public health responses and modeled scenarios for COVID-19 outbreaks caused by SARSCoV-2 assume that infection results in an immune response that protects individuals from future infections or illness for some amount of time. The timescale of protection is a critical determinant of the future impact of the pathogen. The presence or absence of protective immunity due to infection or vaccination (when available) will affect future transmission and illness severity. The dynamics of immunity and nature of protection are relevant to discussions surrounding therapeutic use of convalescent sera as well as efforts to identify individuals with protective immunity. Here, we review the scientific literature on antibody immunity to coronaviruses, including SARS-CoV-2 as well as the related SARS-CoV-1, MERS-CoV and human endemic coronaviruses (HCoVs). We reviewed 1281 abstracts and identified 322 manuscripts relevant to 5 areas of focus: 1) antibody kinetics, 2) correlates of protection, 3) immunopathogenesis, 4) antigenic diversity and cross-reactivity, and 5) population seroprevalence. While studies of SARS-CoV-2 are necessary to determine immune responses to it, evidence from other coronaviruses can provide clues and guide future research.

negative: Synonymous mutations and the molecular evolution of SARS-Cov-2 origins Human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is most closely related, by average genetic distance, to two coronaviruses isolated from bats, RaTG13 and RmYN02. However, there is a segment of high amino acid similarity between human SARS-CoV-2 and a pangolin isolated strain, GD410721, in the receptor binding domain (RBD) of the spike protein, a pattern that can be caused by either recombination or by convergent amino acid evolution driven by natural selection. We perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human SARS-CoV-2 and related strains. We show that the synonymous divergence between the bat derived viruses and SARS-CoV-2 is larger than between GD410721 and SARS-CoV-2 in the RBD, providing strong additional support for the recombination hypothesis. However, the synonymous divergence between pangolin strain and SARS-CoV-2 is also relatively high, which is not consistent with a recent recombination between them, instead it suggests a recombination into RaTG13. We also find a 14-fold increase in the dN/dS ratio from the lineage leading to SARS-CoV-2 to the strains of the current pandemic, suggesting that the vast majority of non-synonymous mutations currently segregating within the human strains have a negative impact on viral fitness. Finally, we estimate that the time to the most recent common ancestor of SARS-CoV-2 and RaTG13 or RmYN02 based on synonymous divergence, is 51.71 years (95% C.I., 28.11-75.31) and 37.02 years (95% C.I., 18.19-55.85), respectively.

negative: Emerging infectious diseases associated with bat viruses Bats play important roles as pollen disseminators and pest predators. However, recent interest has focused on their role as natural reservoirs of pathogens associated with emerging infectious diseases. Prior to the outbreak of severe acute respiratory syndrome (SARS), about 60 bat virus species had been reported. The number of identified bat viruses has dramatically increased since the initial SARS outbreak, and most are putative novel virus species or genotypes. Serious infectious diseases caused by previously identified bat viruses continue to emerge throughout in Asia, Australia, Africa and America. Intriguingly, bats infected by these different viruses seldom display clinical symptoms of illness. The pathogenesis and potential threat of bat-borne viruses to public health remains largely unknown. This review provides a brief overview of bat viruses associated with emerging human infectious diseases.

negative: Complete genome sequence of bat coronavirus HKU2 from Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome Abstract Apart from bat-SARS-CoV, we have identified a novel group 1 coronavirus, bat-CoV HKU2, in Rhinolophus sinicus (Chinese horseshoe bats). Since it has been suggested that the receptor-binding motif (RBM) of SARS-CoV may have been acquired from a group 1 coronavirus, we conducted a surveillance study and identified bat-SARS-CoV and bat-CoV HKU2 in 8.7% and 7.5% respectively of R. sinicus in Hong Kong and Guangdong. Complete genome sequencing of four strains of bat-CoV HKU2 revealed the smallest coronavirus genome (27164 nucleotides) and a unique spike protein evolutionarily distinct from the rest of the genome. This spike protein, sharing similar deletions with other group 2 coronaviruses in its C-terminus, also contained a 15-amino acid peptide homologous to a corresponding peptide within the RBM of spike protein of SARS-CoV, which was absent in other coronaviruses except bat-SARS-CoV. These suggest a common evolutionary origin in the spike protein of bat-CoV HKU2, bat-SARS-CoV, and SARS-CoV.

negative: Nano-ZnO Catalyzed Multicomponent One-Pot Synthesis of Novel Spiro(indoline-pyranodioxine) Derivatives A simple catalytic protocol for the synthesis of novel spiro[indoline-pyranodioxine] derivatives has been developed using ZnO nanoparticle as an efficient, green, and reusable catalyst. The derivatives are obtained in moderate to excellent yield by one-pot three-component reaction of an isatin, malononitrile/ethylcyanoacetate, and 2,2-dimethyl-1,3-dioxane-4,6-dione in absolute ethanol under conventional heating and microwave irradiation. The catalyst was recovered by filtration from the reaction mixture and reused during five consecutive runs without any apparent loss of activity for the same reaction. The mild reaction conditions and recyclability of the catalyst make it environmentally benign synthetic procedure.

negative: Evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of SARS-CoV Abstract The origins and evolutionary history of the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) remain an issue of uncertainty and debate. Based on evolutionary analyses of coronavirus DNA sequences, encompassing an approximately 13kb stretch of the SARS-TOR2 genome, we provide evidence that SARS-CoV has a recombinant history with lineages of types I and III coronavirus. We identified a minimum of five recombinant regions ranging from 83 to 863bp in length and including the polymerase, nsp9, nsp10, and nsp14. Our results are consistent with a hypothesis of viral host jumping events, concomitant with the reassortment of bird and mammalian coronaviruses, a scenario analogous to earlier outbreaks of influenzae.

negative: Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes We aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/µl) and verified using 74 nose and throat swabs. The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript™ III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.

negative: Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in Colombia Introduction: SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemics. The virus crossed the species barrier and established in the human population due to its ability to exploit the ACE receptor for virus entry, which is present and abundant in several tissues, including the lung and respiratory tract, gastrointestinal tract and hearth. Virus interaction with the cellular receptor is mediated by the surface protein, known as Spike. Another structural protein of major importance in the Nucleocapsid, directly interacting with the viral RNA to form the ribonucleocapsid, considered a multifunctional protein, and being the target of the most molecular diagnostics assays. Objective: To describe the frequency of substitutions in spike and nucleocapsid proteins of SARS-CoV-2 circulating in Colombia and evaluate the frequency of these substitutions in SARS-CoV-2 sequences from other countries of South America. Materials and methods: Samples of 43 patients were included for viral RNA detection by real-time RT-PCR using the Charite-Berlin protocol for the amplification of the SARS-CoV-2 E and RdRp genes. Genome sequences were obtained through the Oxford Nanopore and Illumina MiSeq technologies, following the artic.network "nCoV-2019 sequencing protocol". Available genomes were consulted from GISAID, GenBank, and Genome sequence archive (GSA) and a total of 371 genomes sequences from South America were included. The genome sequences were aligned with the Muscle tool using the MEGA X software. Substitution matrices of the Colombian sequences respect to the reference genome (NC_045512) at the nucleotide and amino acid levels were generated for the spike and nucleocapsid gene. Results: substitution D614G in the amino acid sequence of spike protein was found in 86.7% of the Colombian sequences; substitutions G181V and D936Y in 2.3%, respectively. Five substitutions were found in the nucleocapsid protein, with substitution R203K and G204R being the most frequent (13.95 %) in Colombia. The substitutions D614G in Spike and R203K-G204R in nucleocapsid have a frequency of 83% and 28% respectively in sequences from South America. Conclusion: Non-synonymous substitutions were found in the spike and nucleocapsid proteins in Colombian genomes, the most frequent being D614G in Spike and R203K-G204R in nucleocapsid. These substitutions are frequent in the genomes reported for other South American countries. It is necessary to continue with genomic surveillance of the changes in Spike and Nucleocapsid proteins during the SARS-CoV-2 pandemic in Colombia and South America, even more considering that these proteins are the most commonly used antigen in serological tests.

negative: [Evaluation of different genes in the RT-PCR detection of SARS-CoV-2 in respiratory samples and its evolution in infection].

negative: Usefulness of Published PCR Primers in Detecting Human Rhinovirus Infection We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)–specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology.

negative: Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5’UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3’UTR. Finally, five variants destabilize structured elements within the 3’UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.

negative: SARS-CoV-2 Community Transmission During Shelter-in-Place in San Francisco ABSTRACT Background: We characterized SARS-CoV-2 infections in a densely-populated, majority Latinx San Francisco community six-weeks into the city's shelter-in-place order. Methods: We offered SARS-CoV-2 reverse transcription-PCR and antibody (Abbott ARCHITECT IgG) testing, regardless of symptoms, to all residents (>=4 years) and workers in a San Francisco census tract (population: 5,174) at outdoor, community-mobilized events over four days. We estimated SARS-CoV-2 point prevalence (PCR-positive) and cumulative incidence (antibody or PCR-positive) in the census tract and evaluated risk factors for recent (PCR-positive/antibody-negative) versus prior infection (antibody-positive/PCR-negative). SARS-CoV-2 genome recovery and phylogenetics were used to measure viral strain diversity, establish viral lineages present, and estimate number of introductions. Results: We tested 3,953 persons: 40% Latinx; 41% White; 9% Asian/Pacific Islander; and 2% Black. Overall, 2.1% (83/3,871) tested PCR-positive: 95% were Latinx and 52% asymptomatic when tested. 1.7% of residents and 6.0% of workers (non-census tract residents) were PCR-positive. Among 2,598 census tract residents, estimated point prevalence of PCR-positives was 2.3% (95%CI: 1.2-3.8%): 3.9% (95%CI: 2.0-6.4%) among Latinx vs. 0.2% (95%CI: 0.0-0.4%) among non-Latinx persons. Estimated cumulative incidence among residents was 6.1% (95%CI: 4.0-8.6%). Prior infections were 67% Latinx, 16% White, and 17% other ethnicities. Among recent infections, 96% were Latinx. Risk factors for recent infection were Latinx ethnicity, inability to shelter-in-place and maintain income, frontline service work, unemployment, and household income <$50,000/year. Five SARS-CoV-2 phylogenetic lineages were detected. Conclusion: SARS-CoV-2 infections from diverse lineages continued circulating among low-income, Latinx persons unable to work from home and maintain income during San Francisco's shelter-in-place ordinance.

negative: Coronavirus phylogeny based on triplets of nucleic acids bases Abstract We considered the fully overlapping triplets of nucleotide bases and proposed a 2D graphical representation of protein sequences consisting of 20 amino acids and a stop code. Based on this 2D graphical representation, we outlined a new approach to analyze the phylogenetic relationships of coronaviruses by constructing a covariance matrix. The evolutionary distances are obtained through measuring the differences among the two-dimensional curves.

negative: A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2 Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts—based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

negative: Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis of SARS-CoV-2-Related Viruses A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species.IMPORTANCE The ongoing COVID-19 pandemic strongly emphasizes the need for a more complete understanding of the biology and pathogenesis of its causative agent SARS-CoV-2. Despite intense scrutiny, several proteins encoded by the genomes of SARS-CoV-2 and other SARS-like coronaviruses remain enigmatic. Moreover, the high infectivity and severity of SARS-CoV-2 in certain individuals make wet-lab studies currently challenging. In this study, we used a series of computational strategies to identify several fast-evolving regions of SARS-CoV-2 proteins which are potentially under host immune pressure. Most notably, the hitherto-uncharacterized protein encoded by ORF8 is one of them. Using sensitive sequence and structural analysis methods, we show that ORF8 and several other proteins from alpha- and beta-coronavirus comprise novel families of immunoglobulin domain proteins, which might function as potential immune modulators to delay or attenuate the host immune response against the viruses.

negative: Current Insight into the Novel Coronavirus Disease 2019 (COVID-19) SARS-CoV-2 is a novel strain of coronavirus that has not been previously identified in humans. It has been declared a pandemic and has infected at least 1,844,683 individuals and caused 117,021 deaths as of 14th April 2020. Transmission among humans occurs via close contact with an infected individual that produces respiratory droplets. Patients have been shown to undergo acute respiratory distress syndrome, which is defined as cytokine storm. The diagnosis relies on detection of nucleic acid, IgG/IgM antibodies, and a chest radiograph of the suspected individuals. The genome of SARS-CoV-2 is similar to other coronaviruses that comprise of ten open reading frames (ORFs). SARS-CoV-2 spike protein exhibits higher affinity to ACE2 receptor as compared with SARS-CoV. Repurposing drugs like favipiravir, remdesivir, chloroquine, and TMPRSS2 protease inhibitors have been shown to be effective for the treatment of COVID-19. Personal protective measures should be followed to prevent SARS-CoV-2 infection. In addition, a clinical trial of SARS-CoV-2 vaccine, mRNA-1273, has been started. This chapter provides a glimpse of advancements made in the area of SARS-CoV-2 infection by proving recent clinical and research trials in the field.

negative: The global and local distribution of RNA structure throughout the SARS-CoV-2 genome SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.

negative: Descriptive distribution and phylogenetic analysis of feline infectious peritonitis virus isolates of Malaysia The descriptive distribution and phylogeny of feline coronaviruses (FCoVs) were studied in cats suspected of having feline infectious peritonitis (FIP) in Malaysia. Ascitic fluids and/or biopsy samples were subjected to a reverse transcription polymerase chain reaction (RT-PCR) targeted for a conserved region of 3'untranslated region (3'UTR) of the FCoV genome. Eighty nine percent of the sampled animals were positive for the presence of FCoV. Among the FCoV positive cats, 80% of cats were males and 64% were below 2 years of age. The FCoV positive cases included 56% domestic short hair (DSH), 40% Persian, and 4% Siamese cats. The nucleotide sequences of 10 selected amplified products from FIP cases were determined. The sequence comparison revealed that the field isolates had 96% homology with a few point mutations. The extent of homology decreased to 93% when compared with reference strains. The overall branching pattern of phylogenetic tree showed two distinct clusters, where all Malaysian isolates fall into one main genetic cluster. These findings provided the first genetic information of FCoV in Malaysia.