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1,700 | Age-Related Onset of Obesity Corresponds with Metabolic Dysregulation and Altered Microglia Morphology in Mice Deficient for Ifitm Proteins | The IfitmDel mouse lacks all five of the Ifitm genes via LoxP deletion. This animal breeds normally with no obvious defect in development. The IfitmDel animals exhibit a steady and significantly enhanced weight gain relative to wild-type controls beginning about three months of age and under normal feeding conditions. The increased weight corresponds with elevated fat mass, and in tolerance tests they are hyporesponsive to insulin but respond normally to glucose. Both young (4 mo) and older (12 mo) IfitmDel mice have enhanced levels of serum leptin suggesting a defect in leptin/leptin receptor signaling. Analysis of the gene expression profiles in the hypothalamus of IfitmDel animals, compared to WT, demonstrated an altered ratio of Pomc and Npy neuropeptide expression, which likely impairs the satiation response of the IfitmDel animal leading to an increased eating behavior. Also elevated in hypothalamus of IfitmDel mice were pro-inflammatory cytokine expression and reduced IL-10. Anatomical analysis of the hypothalamus using immunohistochemistry revealed that microglia exhibit an abnormal morphology in IfitmDel animals and respond abnormally to Poly:IC challenge. These abnormalities extend the phenotype of the IfitmDel mouse beyond abnormal responses to viral challenge to include a metabolic phenotype and weight gain. Further, this novel phenotype for the IfitmDel mouse could be related to abnormal neuropeptide production, inflammatory status and microglia status in the hypothalamus. |
1,701 | Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis) | Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs. |
1,702 | Antiviral Activity and Possible Mechanism of Action of Constituents Identified in Paeonia lactiflora Root toward Human Rhinoviruses | Human rhinoviruses (HRVs) are responsible for more than half of all cases of the common cold and cost billions of USD annually in medical visits and missed school and work. An assessment was made of the antiviral activities and mechanisms of action of paeonol (PA) and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) from Paeonia lactiflora root toward HRV-2 and HRV-4 in MRC5 cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results were compared with those of a reference control ribavirin. Based on 50% inhibitory concentration values, PGG was 13.4 and 18.0 times more active toward HRV-2 (17.89 μM) and HRV-4 (17.33 μM) in MRC5 cells, respectively, than ribavirin. The constituents had relatively high selective index values (3.3–>8.5). The 100 μg/mL PA and 20 μg/mL PGG did not interact with the HRV-4 particles. These constituents inhibited HRV-4 infection only when they were added during the virus inoculation (0 h), the adsorption period of HRVs, but not after 1 h or later. Moreover, the RNA replication levels of HRVs were remarkably reduced in the MRC5 cultures treated with these constituents. These findings suggest that PGG and PA may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule-1 and low-density lipoprotein receptor), inflammatory cytokines (interleukin (IL)-6, IL-8, tumor necrosis factor, interferon beta, and IL-1β), and Toll-like receptor, which resulted in diminishing symptoms induced by HRV. Global efforts to reduce the level of synthetic drugs justify further studies on P. lactiflora root-derived materials as potential anti-HRV products or lead molecules for the prevention or treatment of HRV. |
1,703 | Human alveolar epithelial type II cells in primary culture | Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. |
1,704 | 3D Structure Prediction of Human β1-Adrenergic Receptor via Threading-Based Homology Modeling for Implications in Structure-Based Drug Designing | Dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β(1)-adrenergic receptor (β(1)-AR) signal cascade. The disturbed β(1)-AR function may be based on an elevated sympathetic tone observed in patients with heart failure. Prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. Hence, β(1)-AR is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3D structure of Homo sapiens β(1)-AR (hsβADR1). This study encompasses elucidation of 3D structural and physicochemical properties of hsβADR1 via threading-based homology modeling. Furthermore, the docking performance of several docking programs including Surflex-Dock, FRED, and GOLD were validated by re-docking and cross-docking experiments. GOLD and Surflex-Dock performed best in re-docking and cross docking experiments, respectively. Consequently, Surflex-Dock was used to predict the binding modes of four hsβADR1 agonists. This study provides clear understanding of hsβADR1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein. |
1,705 | The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei | Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type. |
1,706 | Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin | We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies. |
1,707 | 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. |
1,708 | 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. |
1,709 | Phase 1 Study of Pandemic H1 DNA Vaccine in Healthy Adults | BACKGROUND: A novel, swine-origin influenza A (H1N1) virus was detected worldwide in April 2009, and the World Health Organization (WHO) declared a global pandemic that June. DNA vaccine priming improves responses to inactivated influenza vaccines. We describe the rapid production and clinical evaluation of a DNA vaccine encoding the hemagglutinin protein of the 2009 pandemic A/California/04/2009(H1N1) influenza virus, accomplished nearly two months faster than production of A/California/07/2009(H1N1) licensed monovalent inactivated vaccine (MIV). METHODS: 20 subjects received three H1 DNA vaccinations (4 mg intramuscularly with Biojector) at 4-week intervals. Eighteen subjects received an optional boost when the licensed H1N1 MIV became available. The interval between the third H1 DNA injection and MIV boost was 3–17 weeks. Vaccine safety was assessed by clinical observation, laboratory parameters, and 7-day solicited reactogenicity. Antibody responses were assessed by ELISA, HAI and neutralization assays, and T cell responses by ELISpot and flow cytometry. RESULTS: Vaccinations were safe and well-tolerated. As evaluated by HAI, 6/20 developed positive responses at 4 weeks after third DNA injection and 13/18 at 4 weeks after MIV boost. Similar results were detected in neutralization assays. T cell responses were detected after DNA and MIV. The antibody responses were significantly amplified by the MIV boost, however, the boost did not increased T cell responses induced by DNA vaccine. CONCLUSIONS: H1 DNA vaccine was produced quickly, was well-tolerated, and had modest immunogenicity as a single agent. Other HA DNA prime-MIV boost regimens utilizing one DNA prime vaccination and longer boost intervals have shown significant immunogenicity. Rapid and large-scale production of HA DNA vaccines has the potential to contribute to an efficient response against future influenza pandemics. TRIAL REGISTRATION: Clinicaltrials.gov NCT00973895 |
1,710 | Infectious Bursal Disease Virus VP5 Polypeptide: A Phosphoinositide-Binding Protein Required for Efficient Cell-to-Cell Virus Dissemination | Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism. |
1,711 | Herpes Simplex Virus-1 Fine-Tunes Host’s Autophagic Response to Infection: A Comprehensive Analysis in Productive Infection Models | Herpes simplex virus-1 (HSV-1) infection causes severe conditions, with serious complications, including corneal blindness from uncontrolled ocular infections. An important cellular defense mechanism against HSV-1 infection is autophagy. The autophagic response of the host cell was suggested to be regulated by HSV-1. In this study, we performed a detailed analysis of autophagy in multiple HSV-1-targeted cell types, and under various infection conditions that recapitulate a productive infection model. We found that autophagy was slightly inhibited in one cell type, while in other cell types autophagy maintained its basal levels mostly unchanged during productive infection. This study refines the concept of HSV-1-mediated autophagy regulation to imply either inhibition, or prevention of activation, of the innate immune pathway. |
1,712 | Procalcitonin and C-reactive protein cannot differentiate bacterial or viral infection in COPD exacerbation requiring emergency department visits | BACKGROUND: Viral and bacterial infections are the most common causes of chronic obstructive pulmonary disease (COPD) exacerbations. Whether serum inflammatory markers can differentiate bacterial from virus infection in patients with COPD exacerbation requiring emergency department (ED) visits remains controversial. METHODS: Viral culture and polymerase chain reaction (PCR) were used to identify the viruses in the oropharynx of patients with COPD exacerbations. The bacteria were identified by the semiquantitative culture of the expectorated sputum. The peripheral blood white blood cell (WBC) counts, serum C-reactive protein (CRP), procalcitonin (PCT), and clinical symptoms were compared among patients with different types of infections. RESULTS: Viruses were isolated from 16 (22.2%) of the 72 patients enrolled. The most commonly identified viruses were parainfluenza type 3, influenza A, and rhinovirus. A total of 30 (41.7%) patients had positive bacterial cultures, with the most commonly found bacteria being Haemophilus influenzae and Haemophilus parainfluenzae. Five patients (6.9%) had both positive sputum cultures and virus identification. The WBC, CRP, and PCT levels of the bacteria-positive and bacteria-negative groups were not statistically different. Multivariate analysis showed that patients with increased sputum volumes during the COPD exacerbations had higher risks of recurrent exacerbations in the 1-year period following the first exacerbation. CONCLUSION: WBC, CRP, or PCT could not differentiate between bacterial and viral infections in patients with COPD exacerbation requiring ED visits. Those with increased sputum during a COPD exacerbation had higher risks for recurrent exacerbations. |
1,713 | External validation of scores proposed for estimation of survival probability of patients with severe adult respiratory distress syndrome undergoing extracorporeal membrane oxygenation therapy: a retrospective study | INTRODUCTION: This study was designed as an external validation of the recently proposed Predicting Death for Severe ARDS on V-V ECMO (PRESERVE) score, The respiratory extracorporeal membrane oxygenation survival prediction (RESP) score and a scoring system developed for externally retrieved patients on extracorporeal membrane oxygenation (ECMO) at our institution. All scores are proposed for the estimation of survival probability after ECMO treatment for severe adult respiratory distress syndrome. METHODS: Data from 51 patients (2008 to 2013) were analyzed in this retrospective single-center study. A calculation of an adapted PRESERVE score, the RESP score as well as the score developed for externally retrieved ECMO patients was performed. RESULTS: Seventy one percent of patients received veno-venous (v-v) and 29% venous-arterial (v-a) ECMO support during the study period. Overall survival at 6 months was 55%, with a 61% survival rate for v-v cannulated patients and a 40% survival rate for v-a cannulated patients. The PRESERVE score discriminated survivors and non-survivors with an area under the curve of 0.67 (95% CI 0.52 to 0.82, P = 0.03). Analyzing survival prediction according to cannulation modus, the PRESERVE score and the RESP score significantly predicted survival for patients on v-v ECMO with an area under the curve of 0.75 (95% CI 0.57 to 0.92, P = 0.01) and 0.81 (95% CI 0.67 to 0.95, P = 0.035), respectively, while the scoring system developed for externally retrieved ECMO patients failed to predict survival in our study population. All scores failed to predict mortality for patients on v-a ECMO. CONCLUSION: Our single-center validation confirms that the proposed PRESERVE and RESP score predict survival for patients treated with v-v ECMO for severe adult respiratory distress syndrome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-015-0875-z) contains supplementary material, which is available to authorized users. |
1,714 | New global viral threats | Infectious diseases have caused great catastrophes in human history, as in the example of the plague, which wiped out half of the population in Europe in the 14th century. Ebola virus and H7N9 avian influenza virus are 2 lethal pathogens that we have encountered in the second decade of the 21st century. Ebola infection is currently being seen in West Africa, and H7N9 avian flu appears to have settled in Southeast Asia. This article focuses on the current situation and the future prospects of these potential infectious threats to mankind. |
1,715 | Characterization of Plasmodium vivax-associated admissions to reference hospitals in Brazil and India | BACKGROUND: The benign character formerly attributed to Plasmodium vivax infection has been dismantled by the increasing number of reports of severe disease associated with infection with this parasite, prompting the need for more thorough and comprehensive characterization of the spectrum of resulting clinical complications. Endemic areas exhibit wide variations regarding severe disease frequency. This study, conducted simultaneously in Brazil and India, constitutes, to our knowledge, the first multisite study focused on clinical characterization of P. vivax severe disease. METHODS: Patients admitted with P. vivax mono-infection at reference centers in Manaus (Amazon - Brazil) and Bikaner (Rajasthan - India), where P. vivax predominates, were submitted to standard thorough clinical and laboratory evaluations in order to characterize clinical manifestations and identify concurrent co-morbidities. RESULTS: In total, 778 patients (88.0% above 12 years old) were hospitalized at clinical discretion with PCR-confirmed P. vivax mono-infection (316 in Manaus and 462 in Bikaner), of which 197 (25.3%) presented at least one severity criterion as defined by the World Health Organization (2010). Hyperlactatemia, respiratory distress, hypoglycemia, and disseminated intravascular coagulation were more frequent in Manaus. Noteworthy, pregnancy status was associated as a risk factor for severe disease (OR = 2.03; 95% CI = 1.2-3.4; P = 0.007). The overall case fatality rate was 0.3/1,000 cases in Manaus and 6.1/1,000 cases in Bikaner, with all deaths occurring among patients fulfilling at least one severity criterion. Within this subgroup, case fatality rates increased respectively to 7.5% in Manaus and 4.4% in Bikaner. CONCLUSION: P. vivax-associated severity is not negligible, and although lethality observed for complicated cases was similar, the overall fatality rate was about 20-fold higher in India compared to Brazil, highlighting the variability observed in different settings. Our observations highlight that pregnant women and patients with co-morbidities need special attention when infected by this parasite due to higher risk of complications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-015-0302-y) contains supplementary material, which is available to authorized users. |
1,716 | BAP31, a promising target for the immunotherapy of malignant melanomas | PURPOSE: Malignant melanoma’s (MM) incidence is rising faster than that of any other cancer in the US and the overall survival at 5 years is less than 10%. B cell associated protein 31 (BAP31) is overexpressed in most MMs and might be a promising target for immunotherapy of this disease. EXPERIMENTAL DESIGN: Firstly, we investigated the expression profiles of human BAP31 (hBAP31) and mouse BAP31 (mBAP31) in human and mouse normal tissues, respectively. The expression level of hBAP31 in human MMs and mBAP31 in B16 melanoma cells was also analyzed. Then we constructed novel mBAP31 DNA vaccines and tested there ability to stimulate mBAP31-specific immune responses and antitumor immunity in B16 melanoma-bearing mice. RESULTS: For the first time, we found that protein expression of hBAP31 were dramatically upregulated in human MMs when compared with human normal tissues. Predominant protein expression of mBAP31 was found in mouse B16 melanoma cells but not in mouse important organs. When mice were immunized with mBAP31 DNA vaccines, strong cellular response to mBAP31 was observed in the vaccinated mice. CTLs isolated from immunized mice could effectively kill mBAP31-positive target mouse B16 melanoma tumor cells in vitro and vaccination with mBAP31 DNA vaccines had potent anti-tumor activity in therapeutic model using B16 melanoma cells. CONCLUSIONS: These are the first data supporting a vaccine targeting BAP31 that is capable of inducing effective immunity against BAP31-expressing MMs and will be applicable to human MMs and hBAP31 DNA vaccine warrants investigation in human clinical trials. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13046-015-0153-6) contains supplementary material, which is available to authorized users. |
1,717 | Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5 | Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus. |
1,718 | The Importance of Bacterial and Viral Infections Associated with Adult Asthma Exacerbations in Clinical Practice | BACKGROUND: Viral infection is one of the risk factors for asthma exacerbation. However, which pathogens are related to asthma exacerbation in adults remains unclear. OBJECTIVE: The relation between various infections and adult asthma exacerbations was investigated in clinical practice. METHODS: The study subjects included 50 adult inpatients due to asthma exacerbations and 20 stable outpatients for comparison. The pathogens from a nasopharyngeal swab were measured by multiplex PCR analysis. RESULTS: Asthma exacerbations occurred after a common cold in 48 inpatients. The numbers of patients with viral, bacterial, or both infections were 16, 9, and 9, respectively. The dominant viruses were rhinoviruses, respiratory syncytial virus, influenza virus, and metapneumovirus. The major bacteria were S. pneumoniae and H. influenzae. Compared to pathogen-free patients, the patients with pathogens were older and non-atopic and had later onset of disease, lower FeNO levels, lower IgE titers, and a higher incidence of comorbid sinusitis, COPD, or pneumonia. Compared to stable outpatients, asthma exacerbation inpatients had a higher incidence of smoking and comorbid sinusitis, COPD, or pneumonia. Viruses were detected in 50% of stable outpatients, but a higher incidence of rhinovirus, respiratory syncytial virus, and metapneumovirus infections was observed in asthma exacerbation inpatients. H. influenzae was observed in stable asthmatic patients. Other bacteria, especially S. pneumoniae, were important in asthma exacerbation inpatients. CONCLUSION: Viral or bacterial infections were observed in 70% of inpatients with an asthma exacerbation in clinical practice. Infection with S. pneumoniae was related to adult asthma exacerbation. |
1,719 | Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis | Swine influenza virus and Streptococcus suis are two important contributors to the porcine respiratory disease complex, and both have significant economic impacts. Clinically, influenza virus and Streptococcus suis co-infections in pigs are very common, which often contribute to severe pneumonia and can increase the mortality. However, the co-infection pathogenesis in pigs is unclear. In the present study, co-infection experiments were performed using swine H1N1 influenza virus and Streptococcus suis serotype 2 (SS2). The H1N1-SS2 co-infected pigs exhibited more severe clinical symptoms, serious pathological changes, and robust apoptosis of lungs at 6 days post-infection compared with separate H1N1 and SS2 infections. A comprehensive gene expression profiling using a microarray approach was performed to investigate the global host responses of swine lungs against the swine H1N1 infection, SS2 infection, co-infection, and phosphate-buffered saline control. Results showed 457, 411, and 844 differentially expressed genes in the H1N1, SS2, and H1N1-SS2 groups, respectively, compared with the control. Noticeably, genes associated with the immune, inflammatory, and apoptosis responses were highly overexpressed in the co-infected group. Pathway analysis indicated that the cytokine–cytokine receptor interactions, MAPK, toll-like receptor, complement and coagulation cascades, antigen processing and presentation, and apoptosis pathway were significantly regulated in the co-infected group. However, the genes related to these were less regulated in the separate H1N1 and SS2 infection groups. This observation suggested that a certain level of synergy was induced by H1N1 and SS2 co-infection with significantly stronger inflammatory and apoptosis responses, which may lead to more serious respiratory disease syndrome and pulmonary pathological lesion. |
1,720 | A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication | Influenza A virus causes annual epidemics and occasional pandemics in humans. Here, we investigated four members of the fibroblast growth factor receptor (FGFR) family; FGFR1 to 4, and examined their expression patterns in human lung epithelial cells A549 with influenza A virus infection. We identified a functional role of FGFR1 in influenza A/Puerto Rico/8/1934 (PR8) and A/Anhui/01/2005 (H5N1) virus replication. Our results showed that FGFR1 silencing by siRNA interference promoted influenza A/PR8 and H5N1 virus replication in A549 cells, while lentivirus-mediated exogenous FGFR1 expression significantly suppressed influenza A virus replication; however, FGFR4 did not have the same effects. Moreover, FGFR1 phosphorylation levels were downregulated in A549 cells by influenza A virus infection, while the repression of FGFR1 kinase using PD173074, a potent and selective FGFR1 inhibitor, could enhance virus replication. Furthermore, we found that FGFR1 inhibits influenza virus internalization, but not binding, during viral entry. These results suggested that FGFR1 specifically antagonizes influenza A virus replication, probably by blocking viral entry. |
1,721 | A Neuron-Specific Antiviral Mechanism Prevents Lethal Flaviviral Infection of Mosquitoes | Mosquitoes are natural vectors for many etiologic agents of human viral diseases. Mosquito-borne flaviviruses can persistently infect the mosquito central nervous system without causing dramatic pathology or influencing the mosquito behavior and lifespan. The mechanism by which the mosquito nervous system resists flaviviral infection is still largely unknown. Here we report that an Aedes aegypti homologue of the neural factor Hikaru genki (AaHig) efficiently restricts flavivirus infection of the central nervous system. AaHig was predominantly expressed in the mosquito nervous system and localized to the plasma membrane of neural cells. Functional blockade of AaHig enhanced Dengue virus (DENV) and Japanese encephalitis virus (JEV), but not Sindbis virus (SINV), replication in mosquito heads and consequently caused neural apoptosis and a dramatic reduction in the mosquito lifespan. Consistently, delivery of recombinant AaHig to mosquitoes reduced viral infection. Furthermore, the membrane-localized AaHig directly interfaced with a highly conserved motif in the surface envelope proteins of DENV and JEV, and consequently interrupted endocytic viral entry into mosquito cells. Loss of either plasma membrane targeting or virion-binding ability rendered AaHig nonfunctional. Interestingly, Culex pipien pallens Hig also demonstrated a prominent anti-flavivirus activity, suggesting a functionally conserved function for Hig. Our results demonstrate that an evolutionarily conserved antiviral mechanism prevents lethal flaviviral infection of the central nervous system in mosquitoes, and thus may facilitate flaviviral transmission in nature. |
1,722 | Insect-Specific Flaviviruses: A Systematic Review of Their Discovery, Host Range, Mode of Transmission, Superinfection Exclusion Potential and Genomic Organization | There has been a dramatic increase in the number of insect-specific flaviviruses (ISFs) discovered in the last decade. Historically, these viruses have generated limited interest due to their inability to infect vertebrate cells. This viewpoint has changed in recent years because some ISFs have been shown to enhance or suppress the replication of medically important flaviviruses in co-infected mosquito cells. Additionally, comparative studies between ISFs and medically important flaviviruses can provide a unique perspective as to why some flaviviruses possess the ability to infect and cause devastating disease in humans while others do not. ISFs have been isolated exclusively from mosquitoes in nature but the detection of ISF-like sequences in sandflies and chironomids indicates that they may also infect other dipterans. ISFs can be divided into two distinct phylogenetic groups. The first group currently consists of approximately 12 viruses and includes cell fusing agent virus, Kamiti River virus and Culex flavivirus. These viruses are phylogenetically distinct from all other known flaviviruses. The second group, which is apparently not monophyletic, currently consists of nine viruses and includes Chaoyang virus, Nounané virus and Lammi virus. These viruses phylogenetically affiliate with mosquito/vertebrate flaviviruses despite their apparent insect-restricted phenotype. This article provides a review of the discovery, host range, mode of transmission, superinfection exclusion ability and genomic organization of ISFs. This article also attempts to clarify the ISF nomenclature because some of these viruses have been assigned more than one name due to their simultaneous discoveries by independent research groups. |
1,723 | The association of polymorphisms of TLR4 and CD14 genes with susceptibility to sepsis in a Chinese population | BACKGROUND: Sepsis is now the leading cause of death in the non-cardiovascular intensive care unit (ICU). Recent research suggests that sepsis is likely to be due to an interaction between genetic and environmental factors. Genetic mutations of toll-like receptor 4 (TLR4) and cluster of differentiation 14 (CD14) genes are involved in the immune and (or) inflammatory response. These may contribute to the susceptibility to sepsis in patients. This study was designed to evaluate whether the TLR4 and cluster CD14 gene polymorphisms are associated with susceptibility to sepsis. METHODS: The single nucleotide polymorphisms (SNPs) of TLR4 (rs10759932, rs11536889, rs7873784, rs12377632, rs1927907, rs1153879) and CD14 (rs2569190 and rs2563298) in patients with sepsis and control subjects in the Guangxi Province were analyzed by using the polymerase chain reaction-single base extension (PCR-SBE) and DNA sequencing methods. RESULTS: The rs11536889 polymorphism in TLR4 and rs2563298 polymorphism in CD14 were significantly associated with the risk of sepsis when compared to the control group. The frequencies of rs11536889 and rs2563298 polymorphisms in the group with sepsis were higher than that in the control group (OR = 1.430, 95% CI, 1.032-1.981, P<0.05; OR = 2.454, 95% CI, 1.458-4.130, P<0.05, respectively). Followed up haplotype analysis suggested that there were two haplotypes in which increased risk factors for sepsis were indicated. CONCLUSIONS: The rs11536889 polymorphism in TLR4 and rs2563298 polymorphism in CD14, and two haplotypes were associated with increased susceptibility to sepsis. |
1,724 | Pseudotype-Based Neutralization Assays for Influenza: A Systematic Analysis | The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of hemagglutinin (HA) thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D toward the production of a “universal vaccine” has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1–H16). The accurate and sensitive measurement of antibody responses elicited by these “next-generation” influenza vaccines is, however, hampered by the lack of sensitivity of the traditional influenza serological assays HI, single radial hemolysis, and microneutralization. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies. |
1,725 | A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity | We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common “a” determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log(10) higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody. |
1,726 | Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology | Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide. The nucleocapsid (N) protein is a major structural protein of PRRSV. The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection. In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics. This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein. Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology. Serial growth of PRRSV in different concentrations of 3-AB did not yield viruses that were able to grow with wild type kinetics, suggesting that by targeting a cellular protein crucial for virus biology, resistant phenotypes did not emerge. This study provides further evidence that cellular proteins, which are critical for virus biology, can also be targeted to ablate virus growth and provide a high barrier for the emergence of drug resistance. |
1,727 | Distribution of antibodies against influenza virus in pigs from farrow-to-finish farms in Minas Gerais state, Brazil | BACKGROUND: Swine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds. OBJECTIVES: The objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil. METHODS: Thirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain. RESULTS: The percentages of seropositive animals for H1N1pdm09 and H3N2 were 26·23% and 1·57%, respectively, and the percentages of seropositive herds for both viruses were 96·6% and 13·2%, respectively. CONCLUSIONS: The serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks. |
1,728 | Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications | It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence. |
1,729 | Haematology and Plasma Biochemistry of Wild Black Flying-Foxes, (Pteropus alecto) in Queensland, Australia | This paper establishes reference ranges for hematologic and plasma biochemistry values in wild Black flying-foxes (Pteropus alecto) captured in South East Queensland, Australia. Values were found to be consistent with those of other Pteropus species. Four hundred and forty-seven animals were sampled over 12 months and significant differences were found between age, sex, reproductive and body condition cohorts in the sample population. Mean values for each cohort fell within the determined normal adult reference range, with the exception of elevated levels of alkaline phosphatase in juvenile animals. Hematologic and biochemistry parameters of injured animals showed little or no deviation from the normal reference values for minor injuries, while two animals with more severe injury or abscessation showed leucocytosis, anaemia, thrombocytosis, hyperglobulinemia and hypoalbuminemia. |
1,730 | Epidemiology and Virology of Acute Respiratory Infections During the First Year of Life: A Birth Cohort Study in Vietnam | BACKGROUND: Understanding viral etiology and age-specific incidence of acute respiratory infections in infants can help identify risk groups and inform vaccine delivery, but community-based data is lacking from tropical settings. METHODS: One thousand four hundred and seventy-eight infants in urban Ho Chi Minh City and 981 infants in a semi-rural district in southern Vietnam were enrolled at birth and followed to 1 year of age. Acute respiratory infection (ARI) episodes were identified through clinic-based illness surveillance, hospital admissions and self-reports. Nasopharyngeal swabs were collected from infants with respiratory symptoms and tested for 14 respiratory pathogens using multiplex reverse transcription-polymerase chain reaction. RESULTS: Estimated incidence of ARI was 542 and 2691 per 1000 infant-years, and hospitalization rates for ARI were 81 and 138 per 1000 infant-years, in urban and semi-rural cohorts, respectively, from clinic- and hospital-based surveillance. However self-reported ARI episodes were just 1.5-fold higher in the semi-rural versus urban cohort, indicating that part of the urban–rural difference was explained by under-ascertainment in the urban cohort. Incidence was higher in infants ≥6 months of age than <6 months, but this was pathogen-specific. One or more viruses were detected in 53% (urban) and 64% (semi-rural) of samples from outpatients with ARI and in 78% and 66% of samples from hospitalized ARI patients, respectively. The most frequently detected viruses were rhinovirus, respiratory syncytial virus, influenza virus A and bocavirus. ARI-associated hospitalizations were associated with longer stays and more frequent ICU admission than other infections. CONCLUSIONS: ARI is a significant cause of morbidity in Vietnamese infants and influenza virus A is an under-appreciated cause of vaccine-preventable disease and hospitalizations in this tropical setting. Public health strategies to reduce infant ARI incidence and hospitalization rates are needed. |
1,731 | Beyond resistance: social factors in the general public response to pandemic influenza | BACKGROUND: Influencing the general public response to pandemics is a public health priority. There is a prevailing view, however, that the general public is resistant to communications on pandemic influenza and that behavioural responses to the 2009/10 H1N1 pandemic were not sufficient. Using qualitative methods, this paper investigates how members of the general public respond to pandemic influenza and the hygiene, social isolation and other measures proposed by public health. Going beyond the commonly deployed notion that the general public is resistant to public health communications, this paper examines how health individualism, gender and real world constraints enable and limit individual action. METHODS: In-depth interviews (n = 57) and focus groups (ten focus groups; 59 individuals) were conducted with community samples in Melbourne, Sydney and Glasgow. Participants were selected according to maximum variation sampling using purposive criteria, including: 1) pregnancy in 2009/2010; 2) chronic illness; 3) aged 70 years and over; 4) no disclosed health problems. Verbatim transcripts were subjected to inductive, thematic analysis. RESULTS: Respondents did not express resistance to public health communications, but gave insight into how they interpreted and implemented guidance. An individualistic approach to pandemic risk predominated. The uptake of hygiene, social isolation and vaccine strategies was constrained by seeing oneself ‘at risk’ but not ‘a risk’ to others. Gender norms shape how members of the general public enact hygiene and social isolation. Other challenges pertained to over-reliance on perceived remoteness from risk, expectation of recovery from infection and practical constraints on the uptake of vaccination. CONCLUSIONS: Overall, respondents were engaged with public health advice regarding pandemic influenza, indicating that the idea of public resistance has limited explanatory power. Public communications are endorsed, but challenges persist. Individualistic approaches to pandemic risk inhibit acting for the benefit of others and may deepen divisions in the community according to health status. Public communications on pandemics are mediated by gender norms that may overburden women and limit the action of men. Social research on the public response to pandemics needs to focus on the social structures and real world settings and relationships that shape the action of individuals. |
1,732 | Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape | A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular ‘arms races.’ Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2’-5’-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors. |
1,733 | Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype | Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype. |
1,734 | Contact pathway of coagulation and inflammation | The contact system, also named as plasma kallikrein-kinin system, consists of three serine proteinases: coagulation factors XII (FXII) and XI (FXI), and plasma prekallikrein (PK), and the nonenzymatic cofactor high molecular weight kininogen (HK). This system has been investigated actively for more than 50 years. The components of this system and their interactions have been elucidated from in vitro experiments, which indicates that this system is prothrombotic by activating intrinsic pathway, and proinflammatory by producing bioactive peptide bradykinin. Although the activation of the contact system have been implicated in various types of human disease, in only a few instances is its role clearly defined. In the last 10 years, our understanding of the contact system, particularly its biology and (patho)physiology has greatly increased through investigations using gene-modified animal models. In this review we will describe a revitalized view of the contact system as a critical (patho)physiologic mediator of coagulation and inflammation. |
1,735 | Perturbations at the ribosomal genes loci are at the centre of cellular dysfunction and human disease | Ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) drives cell growth and underlies nucleolar structure and function, indirectly coordinating many fundamental cellular processes. The importance of keeping rDNA transcription under tight control is reflected by the fact that deranged Pol I transcription is a feature of cancer and other human disorders. In this review, we discuss multiple aspects of rDNA function including the relationship between Pol I transcription and proliferative capacity, the role of Pol I transcription in mediating nucleolar structure and integrity, and rDNA/nucleolar interactions with the genome and their influence on heterochromatin and global genome stability. Furthermore, we discuss how perturbations in the structure of the rDNA loci might contribute to human disease, in some cases independent of effects on ribosome biogenesis. |
1,736 | 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. |
1,737 | Drak2 Does Not Regulate TGF-β Signaling in T Cells | Drak2 is a serine/threonine kinase expressed highest in T cells and B cells. Drak2(-/-) mice are resistant to autoimmunity in mouse models of type 1 diabetes and multiple sclerosis. Resistance to these diseases occurs, in part, because Drak2 is required for the survival of autoreactive T cells that induce disease. However, the molecular mechanisms by which Drak2 affects T cell survival and autoimmunity are not known. A recent report demonstrated that Drak2 negatively regulated transforming growth factor-β (TGF-β) signaling in tumor cell lines. Thus, increased TGF-β signaling in the absence of Drak2 may contribute to the resistance to autoimmunity in Drak2(-/-) mice. Therefore, we examined if Drak2 functioned as a negative regulator of TGF-β signaling in T cells, and whether the enhanced susceptibility to death of Drak2(-/-) T cells was due to augmented TGF-β signaling. Using several in vitro assays to test TGF-β signaling and T cell function, we found that activation of Smad2 and Smad3, which are downstream of the TGF-β receptor, was similar between wildtype and Drak2(-/-) T cells. Furthermore, TGF-β-mediated effects on naïve T cell proliferation, activated CD8(+) T cell survival, and regulatory T cell induction was similar between wildtype and Drak2(-/-) T cells. Finally, the increased susceptibility to death in the absence of Drak2 was not due to enhanced TGF-β signaling. Together, these data suggest that Drak2 does not function as a negative regulator of TGF-β signaling in primary T cells stimulated in vitro. It is important to investigate and discern potential molecular mechanisms by which Drak2 functions in order to better understand the etiology of autoimmune diseases, as well as to validate the use of Drak2 as a target for therapeutic treatment of these diseases. |
1,738 | Computational Design of Artificial RNA Molecules for Gene Regulation | RNA interference (RNAi) is a powerful tool for the regulation of gene expression. Small exogenous noncoding RNAs (ncRNAs) such as siRNA and shRNA are the active silencing agents, intended to target and cleave complementary mRNAs in a specific way. They are widely and successfully employed in functional studies, and several ongoing and already completed siRNA-based clinical trials suggest encouraging results in the regulation of overexpressed genes in disease. siRNAs share many aspects of their biogenesis and function with miRNAs, small ncRNA molecules transcribed from endogenous genes which are able to repress the expression of target mRNAs by either inhibiting their translation or promoting their degradation. Although siRNA and artificial miRNA molecules can significantly reduce the expression of overexpressed target genes, cancer and other diseases can also be triggered or sustained by upregulated miRNAs. Thus, in the past recent years, molecular tools for miRNA silencing, such as antagomiRs and miRNA sponges, have been developed. These molecules have shown their efficacy in the derepression of genes downregulated by overexpressed miRNAs. In particular, while a single antagomiR is able to inhibit a single complementary miRNA, an artificial sponge construct usually contains one or more binding sites for one or more miRNAs and functions by competing with the natural targets of these miRNAs. As a consequence, natural miRNA targets are reexpressed at their physiological level. In this chapter we review the most successful methods for the computational design of siRNAs, antagomiRs, and miRNA sponges and describe the most popular tools that implement them. |
1,739 | Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4(+) CD25(+) Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice | BACKGROUND: The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4(+)CD25(+) Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS: Key parameters for infection outcome in E. multilocularis-infected fgl2(-/-) (AE-fgl2(-/-)) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4(+)CD25(+) Treg suspensions were incubated with CD4(+) effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2(-/-) mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS: FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases. |
1,740 | West Nile Virus Positive Blood Donation and Subsequent Entomological Investigation, Austria, 2014 | The detection of West Nile virus (WNV) nucleic acid in a blood donation from Vienna, Austria, as well as in Culex pipiens pupae and egg rafts, sampled close to the donor’s residence, is reported. Complete genomic sequences of the human- and mosquito-derived viruses were established, genetically compared and phylogenetically analyzed. The viruses were not identical, but closely related to each other and to recent Czech and Italian isolates, indicating co-circulation of related WNV strains within a confined geographic area. The detection of WNV in a blood donation originating from an area with low WNV prevalence in humans (only three serologically diagnosed cases between 2008 and 2014) is surprising and emphasizes the importance of WNV nucleic acid testing of blood donations even in such areas, along with active mosquito surveillance programs. |
1,741 | Detection of antibodies against avian influenza virus by protein microarray using nucleoprotein expressed in insect cells | Avian influenza (AI) is an infectious disease caused by avian influenza viruses (AIVs) which belong to the influenza virus A group. AI causes tremendous economic losses in poultry industry and pose great threatens to human health. Active serologic surveillance is necessary to prevent and control the spread of AI. In this study, a protein microarray using nucleoprotein (NP) of H5N1 AIV expressed in insect cells was developed to detect antibodies against AIV NP protein. The protein microarray was used to test Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), AIV positive and negative sera. The results indicated that the protein microarray could hybridize specifically with antibodies against AIV with strong signals and without cross-hybridization. Moreover, 76 field serum samples were detected by microarray, enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). The positive rate was 92.1% (70/76), 93.4% (71/76) and 89.4% (68/76) by protein microarray, ELISA and HI test, respectively. Compared with ELISA, the microarray showed 100% (20/20) agreement ratio in chicken and 98.2% (55/56) in ornamental bird. In conclusion, this method provides an alternative serological diagnosis for influenza antibody screening and will provide a basis for the development of protein microarrays that can be used to respectively detect antibodies of different AIV subtypes and other pathogens. |
1,742 | Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-γ Stimulated HaCaT Human Keratinocytes | Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation. |
1,743 | Prevalence and Incidence of Respiratory Syncytial Virus and Other Respiratory Viral Infections in Children Aged 6 Months to 10 Years With Influenza-like Illness Enrolled in a Randomized Trial | Background. The high burden of respiratory syncytial virus (RSV)-associated morbidity and mortality makes vaccine development a priority. Methods. As part of an efficacy trial of pandemic influenza vaccines (NCT01051661), RSV epidemiology in healthy children aged 6 months to <10 years at first vaccination with influenza-like illness (ILI) was evaluated in Australia, Brazil, Colombia, Costa Rica, Mexico, the Philippines, Singapore, and Thailand between February 2010 and August 2011. Active surveillance for ILI was conducted for approximately 1 year, with nasal and throat swabs analyzed by polymerase chain reaction. The prevalence and incidence of RSV among ILI episodes were calculated. Results. A total of 6266 children were included, of whom 2421 experienced 3717 ILI episodes with a respiratory sample available. RSV was detected for 359 ILI episodes, a prevalence of 9.7% (95% confidence interval: 8.7–10.7). The highest prevalence was in children aged 12–23 or 24–35 months in all countries except the Philippines, where it was in children aged 6–11 months. The incidence of RSV-associated ILI was 7.0 (6.3–7.7) per 100 person-years (PY). Eighty-eight ILI episodes resulted in hospitalization, of which 8 were associated with RSV (prevalence 9.1% [4.0–17.1]; incidence 0.2 [0.1–0.3] per 100 PY). The incidence of RSV-associated ILI resulting in medical attendance was 6.0 (5.4–6.7) per 100 PY. RSV B subtypes were observed more frequently than A subtypes. Conclusions. Active surveillance demonstrated the considerable burden of RSV-associated illness that would not be identified through hospital-based surveillance, with a substantial part of the burden occurring in older infants and children. |
1,744 | Environmental Conditions Affect Exhalation of H3N2 Seasonal and Variant Influenza Viruses and Respiratory Droplet Transmission in Ferrets | The seasonality of influenza virus infections in temperate climates and the role of environmental conditions like temperature and humidity in the transmission of influenza virus through the air are not well understood. Using ferrets housed at four different environmental conditions, we evaluated the respiratory droplet transmission of two influenza viruses (a seasonal H3N2 virus and an H3N2 variant virus, the etiologic virus of a swine to human summertime infection) and concurrently characterized the aerosol shedding profiles of infected animals. Comparisons were made among the different temperature and humidity conditions and between the two viruses to determine if the H3N2 variant virus exhibited enhanced capabilities that may have contributed to the infections occurring in the summer. We report here that although increased levels of H3N2 variant virus were found in ferret nasal wash and exhaled aerosol samples compared to the seasonal H3N2 virus, enhanced respiratory droplet transmission was not observed under any of the environmental settings. However, overall environmental conditions were shown to modulate the frequency of influenza virus transmission through the air. Transmission occurred most frequently at 23°C/30%RH, while the levels of infectious virus in aerosols exhaled by infected ferrets agree with these results. Improving our understanding of how environmental conditions affect influenza virus infectivity and transmission may reveal ways to better protect the public against influenza virus infections. |
1,745 | Forecasting the 2013–2014 Influenza Season Using Wikipedia | Infectious diseases are one of the leading causes of morbidity and mortality around the world; thus, forecasting their impact is crucial for planning an effective response strategy. According to the Centers for Disease Control and Prevention (CDC), seasonal influenza affects 5% to 20% of the U.S. population and causes major economic impacts resulting from hospitalization and absenteeism. Understanding influenza dynamics and forecasting its impact is fundamental for developing prevention and mitigation strategies. We combine modern data assimilation methods with Wikipedia access logs and CDC influenza-like illness (ILI) reports to create a weekly forecast for seasonal influenza. The methods are applied to the 2013-2014 influenza season but are sufficiently general to forecast any disease outbreak, given incidence or case count data. We adjust the initialization and parametrization of a disease model and show that this allows us to determine systematic model bias. In addition, we provide a way to determine where the model diverges from observation and evaluate forecast accuracy. Wikipedia article access logs are shown to be highly correlated with historical ILI records and allow for accurate prediction of ILI data several weeks before it becomes available. The results show that prior to the peak of the flu season, our forecasting method produced 50% and 95% credible intervals for the 2013-2014 ILI observations that contained the actual observations for most weeks in the forecast. However, since our model does not account for re-infection or multiple strains of influenza, the tail of the epidemic is not predicted well after the peak of flu season has passed. |
1,746 | Transcriptional Profiling of Host Gene Expression in Chicken Embryo Fibroblasts Infected with Reticuloendotheliosis Virus Strain HA1101 | Reticuloendotheliosis virus (REV), a member of the Gammaretrovirus genus in the Retroviridae family, causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. To better understand the host interactions at the transcriptional level, microarray data analysis was performed in chicken embryo fibroblast cells at 1, 3, 5, and 7 days after infection with REV. This study identified 1,785 differentially expressed genes that were classified into several functional groups including signal transduction, immune response, biological adhesion and endocytosis. Significant differences were mainly observed in the expression of genes involved in the immune response, especially during the later post-infection time points. These results revealed that differentially expressed genes IL6, STAT1, MyD88, TLRs, NF-κB, IRF-7, and ISGs play important roles in the pathogenicity of REV infection. Our study is the first to use microarray analysis to investigate REV, and these findings provide insights into the underlying mechanisms of the host antiviral response and the molecular basis of viral pathogenesis. |
1,747 | Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs | Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment. |
1,748 | Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice | Dickkopf-1 (DKK1), a secretory inhibitor of canonical Wnt signaling, plays a critical role in certain bone loss diseases. Studies have shown that serum levels of DKK1 are significantly higher in rheumatoid arthritis (RA) patients and are correlated with the severity of the disease, which indicates the possibility that bone erosion in RA may be inhibited by neutralizing the biological activity of DKK1. In this study, we selected a panel of twelve peptides using the software DNASTAR 7.1 and screened high affinity and immunogenicity epitopes in vitro and in vivo assays. Furthermore, we optimized four B cell epitopes to design a novel DKK1 multiepitope DNA vaccine and evaluated its bone protective effects in collagen-induced arthritis (CIA), a mouse model of RA. High level expression of the designed vaccine was measured in supernatant of COS7 cells. In addition, intramuscular immunization of BALB/c mice with this vaccine was also highly expressed and sufficient to induce the production of long-term IgG, which neutralized natural DKK1 in vivo. Importantly, this vaccine significantly attenuated bone erosion in CIA mice compared with positive control mice. These results provide evidence for the development of a DNA vaccine targeted against DKK1 to attenuate bone erosion. |
1,749 | A returning migrant worker with avian influenza A (H7N9) virus infection in Guizhou, China: a case report | INTRODUCTION: Human infection with avian influenza A (H7N9) virus was first reported on March, 2013 in the Yangtze River Delta region of China. The majority of human cases were detected in mainland China; other regions out of mainland China reported imported human cases, including Hong Kong SAR, Taiwan (the Republic of China) and Malaysia, due to human transportation. Here, we report the first human case of H7N9 infection imported into Guizhou Province during the Spring Festival travel season in January 2014. CASE PRESENTATION: In early January 2014, a 38-year-old healthy Chinese man, a migrant worker returning from previously H7N9-affected Zhejiang Province, was identified as the first human case of infection with avian influenza A(H7N9) virus in Guizhou Province. He developed fever in Zhejiang at the beginning of January 2014, and returned to Guizhou for the Chinese New Year the next day. He went to seek medical care, but deteriorated rapidly and died on day 8 after his illness onset. The influenza virus A/Guizhou/01502/2014 isolated from the patient had 99% identity with viruses circulating in the Yangtze River Delta region. Selected amino acids substitutions, well-known to be associated with mammalian adaptation, viral replication and drug resistance were similar to other H7N9 viruses circulating in humans. CONCLUSIONS: Epidemiology investigation and laboratory results confirmed it was the first imported case of H7N9 infection in Guizhou Province. This finding further indicated that more human H7N9 cases may be detected in other regions due to frequent travel both domestically and internationally. |
1,750 | Statin Treatment and Mortality: Propensity Score-Matched Analyses of 2007–2008 and 2009–2010 Laboratory-Confirmed Influenza Hospitalizations | Background. Annual influenza epidemics are responsible for substantial morbidity and mortality. The use of immunomodulatory agents such as statins to target host inflammatory responses in influenza virus infection has been suggested as an adjunct treatment, especially during pandemics, when antiviral quantities are limited or vaccine production can be delayed. Methods. We used population-based, influenza hospitalization surveillance data, propensity score-matched analysis, and Cox regression to determine whether there was an association between mortality (within 30 days of a positive influenza test) and statin treatment among hospitalized cohorts from 2 influenza seasons (October 1, 2007 to April 30, 2008 and September 1, 2009 to April 31, 2010). Results. Hazard ratios for death within the 30-day follow-up period were 0.41 (95% confidence interval [CI], .25–.68) for a matched sample from the 2007–2008 season and 0.77 (95% CI, .43–1.36) for a matched sample from the 2009 pandemic. Conclusions. The analysis suggests a protective effect against death from influenza among patients hospitalized in 2007–2008 but not during the pandemic. Sensitivity analysis indicates the findings for 2007–2008 may be influenced by unmeasured confounders. This analysis does not support using statins as an adjunct treatment for preventing death among persons hospitalized for influenza. |
1,751 | Widespread Recombination, Reassortment, and Transmission of Unbalanced Compound Viral Genotypes in Natural Arenavirus Infections | Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential. |
1,752 | Detection of Acute HIV-1 Infection by RT-LAMP | A rapid, cost-effective diagnostic test for the detection of acute HIV-1 infection is highly desired. Isothermal amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for the development of a rapid nucleic acid amplification test (NAAT) because they are quick, easy to perform and do not require complex, dedicated equipment and laboratory space. In this study, we assessed the ability of the HIV-1 RT-LAMP assay to detect acute HIV infection as compared to a representative rapid antibody test and several FDA-approved laboratory-based assays. The HIV-1 RT-LAMP assay detected seroconverting individuals one to three weeks earlier than a rapid HIV antibody test and up to two weeks earlier than a lab-based antigen/antibody (Ag/Ab) combo enzyme immunoassay (EIA). RT-LAMP was not as sensitive as a lab-based qualitative RNA assay, which could be attributed to the significantly smaller nucleic acid input volume. To our knowledge, this is the first demonstration of detecting acute HIV infection using the RT-LAMP assay. The availability of a rapid NAAT, such as the HIV-1 RT-LAMP assay, at the point of care (POC) or in laboratories that do not have access to large platform NAAT could increase the percentage of individuals who receive an acute HIV infection status or confirmation of their HIV status, while immediately linking them to counseling and medical care. In addition, early knowledge of HIV status could lead to reduced high-risk behavior at a time when individuals are at a higher risk for transmitting the virus. |
1,753 | Influenza A Virus on Oceanic Islands: Host and Viral Diversity in Seabirds in the Western Indian Ocean | Ducks and seabirds are natural hosts for influenza A viruses (IAV). On oceanic islands, the ecology of IAV could be affected by the relative diversity, abundance and density of seabirds and ducks. Seabirds are the most abundant and widespread avifauna in the Western Indian Ocean and, in this region, oceanic islands represent major breeding sites for a large diversity of potential IAV host species. Based on serological assays, we assessed the host range of IAV and the virus subtype diversity in terns of the islands of the Western Indian Ocean. We further investigated the spatial variation in virus transmission patterns between islands and identified the origin of circulating viruses using a molecular approach. Our findings indicate that terns represent a major host for IAV on oceanic islands, not only for seabird-related virus subtypes such as H16, but also for those commonly isolated in wild and domestic ducks (H3, H6, H9, H12 subtypes). We also identified strong species-associated variation in virus exposure that may be associated to differences in the ecology and behaviour of terns. We discuss the role of tern migrations in the spread of viruses to and between oceanic islands, in particular for the H2 and H9 IAV subtypes. |
1,754 | Complete Genome Sequence of a Porcine Epidemic Diarrhea Virus from a Novel Outbreak in Belgium, January 2015 | Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. Here, we report the complete genome sequence (28,028 nt) of a PEDV strain isolated during a novel outbreak in Belgium. |
1,755 | Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium | Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections. |
1,756 | Homology-Independent Metrics for Comparative Genomics | A mainstream procedure to analyze the wealth of genomic data available nowadays is the detection of homologous regions shared across genomes, followed by the extraction of biological information from the patterns of conservation and variation observed in such regions. Although of pivotal importance, comparative genomic procedures that rely on homology inference are obviously not applicable if no homologous regions are detectable. This fact excludes a considerable portion of “genomic dark matter” with no significant similarity — and, consequently, no inferred homology to any other known sequence — from several downstream comparative genomic methods. In this review we compile several sequence metrics that do not rely on homology inference and can be used to compare nucleotide sequences and extract biologically meaningful information from them. These metrics comprise several compositional parameters calculated from sequence data alone, such as GC content, dinucleotide odds ratio, and several codon bias metrics. They also share other interesting properties, such as pervasiveness (patterns persist on smaller scales) and phylogenetic signal. We also cite examples where these homology-independent metrics have been successfully applied to support several bioinformatics challenges, such as taxonomic classification of biological sequences without homology inference. They where also used to detect higher-order patterns of interactions in biological systems, ranging from detecting coevolutionary trends between the genomes of viruses and their hosts to characterization of gene pools of entire microbial communities. We argue that, if correctly understood and applied, homology-independent metrics can add important layers of biological information in comparative genomic studies without prior homology inference. |
1,757 | Diffuse alveolar damage associated mortality in selected acute respiratory distress syndrome patients with open lung biopsy | INTRODUCTION: Diffuse alveolar damage (DAD) is the pathological hallmark of acute respiratory distress syndrome (ARDS), however, the presence of DAD in the clinical criteria of ARDS patients by Berlin definition is little known. This study is designed to investigate the role of DAD in ARDS patients who underwent open lung biopsy. METHODS: We retrospectively reviewed all ARDS patients who met the Berlin definition and underwent open lung biopsy from January 1999 to January 2014 in a referred medical center. DAD is characterized by hyaline membrane formation, lung edema, inflammation, hemorrhage and alveolar epithelial cell injury. Clinical data including baseline characteristics, severity of ARDS, clinical and pathological diagnoses, and survival outcomes were analyzed. RESULTS: A total of 1838 patients with ARDS were identified and open lung biopsies were performed on 101 patients (5.5 %) during the study period. Of these 101 patients, the severity of ARDS on diagnosis was mild of 16.8 %, moderate of 56.5 % and severe of 26.7 %. The hospital mortality rate was not significant difference between the three groups (64.7 % vs 61.4 % vs 55.6 %, p = 0.81). Of the 101 clinical ARDS patients with open lung biopsies, 56.4 % (57/101) patients had DAD according to biopsy results. The proportion of DAD were 76.5 % (13/17) in mild, 56.1 % (32/57) in moderate and 44.4 % (12/27) in severe ARDS and there is no significant difference between the three groups (p = 0.113). Pathological findings of DAD patients had a higher hospital mortality rate than non-DAD patients (71.9 % vs 45.5 %, p = 0.007). Pathological findings of DAD (odds ratio: 3.554, 95 % CI, 1.385–9.12; p = 0.008) and Sequential Organ Failure Assessment score on the biopsy day (odds ratio: 1.424, 95 % CI, 1.187–1.707; p<0.001) were significantly and independently associated with hospital mortality. The baseline demographics and clinical characteristics were not significantly different between DAD and non-DAD patients. CONCLUSIONS: The correlation of pathological findings of DAD and ARDS diagnosed by Berlin definition is modest. A pathological finding of DAD in ARDS patients is associated with hospital mortality and there are no clinical characteristics that could identify DAD patients before open lung biopsy. |
1,758 | Influenza induces IL-8 and GM-CSF secretion by human alveolar epithelial cells through HGF/c-Met and TGF-α/EGFR signaling | The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E(2) during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections. |
1,759 | Computational Docking Study of p7 Ion Channel from HCV Genotype 3 and Genotype 4 and Its Interaction with Natural Compounds | BACKGROUND: The current standard care therapy for hepatitis C virus (HCV) infection consists of two regimes, namely interferon-based and interferon-free treatments. The treatment through the combination of ribavirin and pegylated interferon is expensive, only mildly effective, and is associated with severe side effects. In 2011, two direct-acting antiviral (DAA) drugs, boceprevir and telaprevir, were licensed that have shown enhanced sustained virologic response (SVR) in phase III clinical trial, however, these interferon-free treatments are more sensitive to HCV genotype 1 infection. The variable nature of HCV, and the limited number of inhibitors developed thus aim in expanding the repertoire of available drug targets, resulting in targeting the virus assembly therapeutically. AIM: We conducted this study to predict the 3D structure of the p7 protein from the HCV genotypes 3 and 4. Approximately 63 amino acid residues encoded in HCV render this channel sensitive to inhibitors, making p7 a promising target for novel therapies. HCV p7 protein forms a small membrane known as viroporin, and is essential for effective self-assembly of large channels that conduct cation assembly and discharge infectious virion particles. METHOD: In this study, we screened drugs and flavonoids known to disrupt translation and production of HCV proteins, targeted against the active site of p7 residues of HCV genotype 3 (GT3) (isolatek3a) and HCV genotype 4a (GT4) (isolateED43). Furthermore, we conducted a quantitative structure–activity relationship and docking interaction study. RESULTS: The drug NB-DNJ formed the highest number of hydrogen bond interactions with both modeled p7 proteins with high interaction energy, followed by BIT225. A flavonoid screen demonstrated that Epigallocatechin gallate (EGCG), nobiletin, and quercetin, have more binding modes in GT3 than in GT4. Thus, the predicted p7 protein molecule of HCV from GT3 and GT4 provides a general avenue to target structure-based antiviral compounds. CONCLUSIONS: We hypothesize that the inhibitors of viral p7 identified in this screen may be a new class of potent agents, but further confirmation in vitro and in vivo is essential. This structure-guided drug design for both GT3 and GT4 can lead to the identification of drug-like natural compounds, confirming p7 as a new target in the rapidly increasing era of HCV. |
1,760 | T(FH) cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1 | CD4(+) T follicular helper cells (T(FH)) in germinal centers are required for maturation of B-cells. While the role of T(FH)-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of T(FH) (CXCR5(+)PD-1(++)) and precursor-T(FH) (CXCR5(+)PD-1(+)) cells. The majority of T(FH)-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of T(FH)-cells mainly in the Peyer’s patches and FRT. The novel finding of T(FH)-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study T(FH)-cells and to evaluate HIV-1 vaccines. |
1,761 | Ebola Risk Perception in Germany, 2014 | Ebola virus disease (EVD) outbreaks have occurred during the past 5 decades, but none has affected European countries like the 2014 epidemic in West Africa. We used an online questionnaire to investigate risk perceptions in Germany during this epidemic peak. Our questionnaire covered risk perceptions, knowledge about transmission routes, media use, reactions to the outbreak, attitudes toward measures to prevent the spread of EVD and vaccination against EVD, and willingness to volunteer for aid missions. Of 974 participants, 29% indicated that they worried about EVD, 4% correctly stated virus transmission routes, and 75% incorrectly rated airborne transmission and transmission by asymptomatic patients as possible. Many indicated that if a patient were flown to Germany for treatment in a nearby hospital, they would adapt preventive behavior. Although most participants were not worried about EVD at the current stage of the epidemic, misperceptions regarding transmission were common and could trigger inappropriate behavior changes. |
1,762 | Pneumonia Outbreak Caused by Chlamydophila pneumoniae among US Air Force Academy Cadets, Colorado, USA | During October 2013–May 2014, there were 102 cases of pneumonia diagnosed in US Air Force Academy cadets. A total of 73% of tested nasal washes contained Chlamydophila pneumoniae. This agent can be considered to be present on campus settings during outbreaks with numerous, seemingly disconnected cases of relatively mild pneumonia. |
1,763 | Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment | In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. |
1,764 | Interaction between the Natural Components in Danhong Injection (DHI) with Serum Albumin (SA) and the Influence of the Coexisting Multi-Components on the SaB-BSA Binding System: Fluorescence and Molecular Docking Studies | Danhong injection (DHI) is a widely used Chinese Materia Medica standardized product for the clinical treatment of ischemic encephalopathy and coronary heart disease. The bindings of eight natural components in DHI between bovine serum albumin (BSA) were studied by fluorescence spectroscopy technology and molecular docking. According to the results, the quenching process of salvianolic acid B and hydroxysafflor yellow A was a static quenching procedure through the analysis of quenching data by the Stern-Volmer equation, the modified Stern-Volmer equation, and the modified Scatchard equation. Meanwhile, syringin (Syr) enhanced the fluorescence of BSA, and the data were analyzed using the Lineweaver-Burk equation. Molecular docking suggested that all of these natural components bind to serum albumin at the site I location. Further competitive experiments of SaB confirmed the result of molecular docking studies duo to the displacement of warfarin by SaB. Base on these studies, we selected SaB as a research target because it presented the strongest binding ability to BSA and investigated the influence of the multi-components coexisting in DHI on the interaction between the components of the SaB-BSA binding system. The participation of these natural components in DHI affected the interaction between the components of the SaB-BSA system. Therefore, when DHI is used in mammals, SaB is released from serum albumin more quickly than it is used alone. This work would provide a new experiment basis for revealing the scientific principle of compatibility for Traditional Chinese Medicine. |
1,765 | IFN-λ: A New Class of Interferon with Distinct Functions-Implications for Hepatitis C Virus Research | Pegylated interferon-α and ribavirin (PEG-IFN/RBV) is widely used to treat chronic hepatitis C virus infection with notorious adverse reactions since the broad expression of IFN-α receptors on all nucleated cells. Accordingly, a Type III IFN with restricted receptors distribution is much safer as an alternative for HCV therapy. In addition, single nucleotide polymorphisms (SNPs) near the human IFN-λ3 gene, IL-28B, correlate strongly with the ability to achieve a sustained virological response (SVR) to therapy with pegylated IFN-α plus ribavirin in patients infected with chronic hepatitis C. Furthermore, we also discuss the most recent findings: IFN-λ4 predicts treatment outcomes of HCV infection. In consideration of the apparent limitations of current HCV therapy, especially high failure rate and universal side effects, prediction of treatment outcomes prior to the initiation of treatment and developing new alternative drugs are two important goals in HCV research. |
1,766 | Human resource crises in German hospitals—an explorative study | BACKGROUND: The complexity of providing medical care in a high-tech environment with a highly specialized, limited labour force makes hospitals more crisis-prone than other industries. An effective defence against crises is only possible if the organizational resilience and the capacity to handle crises become part of the hospitals’ organizational culture. To become more resilient to crises, a raised awareness—especially in the area of human resource (HR)—is necessary. The aim of this paper is to contribute to the process robustness against crises through the identification and evaluation of relevant HR crises and their causations in hospitals. METHODS: Qualitative and quantitative methods were combined to identify and evaluate crises in hospitals in the HR sector. A structured workshop with experts was conducted to identify HR crises and their descriptions, as well as causes and consequences for patients and hospitals. To evaluate the findings, an online survey was carried out to rate the occurrence (past, future) and dangerousness of each crisis. RESULTS: Six HR crises were identified in this study: staff shortages, acute loss of personnel following a pandemic, damage to reputation, insufficient communication during restructuring, bullying, and misuse of drugs. The highest occurrence probability in the future was seen in staff shortages, followed by acute loss of personnel following a pandemic. Staff shortages, damage to reputation, and acute loss of personnel following a pandemic were seen as the most dangerous crises. CONCLUSIONS: The study concludes that coping with HR crises in hospitals is existential for hospitals and requires increased awareness. The six HR crises identified occurred regularly in German hospitals in the past, and their occurrence probability for the future was rated as high. |
1,767 | Surveillance of Acute Respiratory Infections Using Community-Submitted Symptoms and Specimens for Molecular Diagnostic Testing | Participatory systems for surveillance of acute respiratory infection give real-time information about infections circulating in the community, yet to-date are limited to self-reported syndromic information only and lacking methods of linking symptom reports to infection types. We developed the GoViral platform to evaluate whether a cohort of lay volunteers could, and would find it useful to, contribute self-reported symptoms online and to compare specimen types for self-collected diagnostic information of sufficient quality for respiratory infection surveillance. Volunteers were recruited, given a kit (collection materials and customized instructions), instructed to report their symptoms weekly, and when sick with cold or flu-like symptoms, requested to collect specimens (saliva and nasal swab). We compared specimen types for respiratory virus detection sensitivity (via polymerase-chain-reaction) and ease of collection. Participants were surveyed to determine receptivity to participating when sick, to receiving information on the type of pathogen causing their infection and types circulating near them. Between December 1 2013 and March 1 2014, 295 participants enrolled in the study and received a kit. Of those who reported symptoms, half (71) collected and sent specimens for analysis. Participants submitted kits on average 2.30 days (95 CI: 1.65 to 2.96) after symptoms began. We found good concordance between nasal and saliva specimens for multiple pathogens, with few discrepancies. Individuals report that saliva collection is easiest and report that receiving information about what pathogen they, and those near them, have is valued and can shape public health behaviors. Community-submitted specimens can be used for the detection of acute respiratory infection with individuals showing receptivity for participating and interest in a real-time picture of respiratory pathogens near them. |
1,768 | Alternative Antigen Processing for MHC Class I: Multiple Roads Lead to Rome | The well described conventional antigen-processing pathway is accountable for most peptides that end up in MHC class I molecules at the cell surface. These peptides experienced liberation by the proteasome and transport by the peptide transporter TAP. However, there are multiple roads that lead to Rome, illustrated by the increasing number of alternative processing pathways that have been reported during last years. Interestingly, TAP-deficient individuals do not succumb to viral infections, suggesting that CD8 T cell immunity is sufficiently supported by alternative TAP-independent processing pathways. To date, a diversity of viral and endogenous TAP-independent peptides have been identified in the grooves of different MHC class I alleles. Some of these peptides are not displayed by normal TAP-positive cells and we therefore called them TEIPP, for “T-cell epitopes associated with impaired peptide processing.” TEIPPs are hidden self-antigens, are derived from normal housekeeping proteins, and are processed via unconventional processing pathways. Per definition, TEIPPs are presented via TAP-independent pathways, but recent data suggest that part of this repertoire still depend on proteasome and metalloprotease activity. An exception is the C-terminal peptide of the endoplasmic reticulum (ER)-membrane-spanning ceramide synthase Trh4 that is surprisingly liberated by the signal peptide peptidase (SPP), the proteolytic enzyme involved in cleaving leader sequences. The intramembrane cleaving SPP is thereby an important contributor of TAP-independent peptides. Its family members, like the Alzheimer’s related presenilins, might contribute as well, according to our preliminary data. Finally, alternative peptide routing is an emerging field and includes processes like the unfolded protein response, the ER-associated degradation, and autophagy-associated vesicular pathways. These data convince us that there is a world to be discovered in the field of unconventional antigen processing. |
1,769 | The Tie2-agonist Vasculotide rescues mice from influenza virus infection | Seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. Antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. Thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. Severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. We hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. Here we demonstrate that the Tie2-agonist tetrameric peptide Vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. The effect required Tie2, was independent of viral replication and did not impair lung neutrophil recruitment. Administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, Vasculotide is inexpensive to produce, is chemically stable and is unrelated to any Tie2 ligands. Thus, Vasculotide may represent a novel and practical therapy for severe infections with influenza. |
1,770 | An Ultrasensitive Mechanism Regulates Influenza Virus-Induced Inflammation | Influenza viruses present major challenges to public health, evident by the 2009 influenza pandemic. Highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. However, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. Here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. We found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. Cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin 6 and monocyte chemotactic protein 1, exhibit ultrasensitive behavior. A systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the Toll-like receptor pathway that regulates STAT1 phosphorylation. This study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. The approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. |
1,771 | Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion | Adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. There are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. The first includes an insertion of the foreign gene expression cassette into the E1 region. The second strategy is antigen incorporation into the viral capsid proteins. To extend this methodology, we have searched for new sites at the human adenovirus serotype 5 major capsid protein hexon for a vaccine antigen insertion. To this end, we utilized sites in the hexon hypervariable region (HVR) 7, 8 and 9 to display a 15-mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. However, we could not rescue the viruses with the insertions of the peptide into HVR 8 and 9, consistent with the viruses being unable to tolerate insertions at these sites. In contrast, the virus with the insertion of the peptide in HVR 7 was viable - growing well in cell culture and the inserted peptide was exposed on the virion surface. |
1,772 | The venomous cocktail of the vampire snail Colubraria reticulata (Mollusca, Gastropoda) | BACKGROUND: Hematophagy arose independently multiple times during metazoan evolution, with several lineages of vampire animals particularly diversified in invertebrates. However, the biochemistry of hematophagy has been studied in a few species of direct medical interest and is still underdeveloped in most invertebrates, as in general is the study of venom toxins. In cone snails, leeches, arthropods and snakes, the strong target specificity of venom toxins uniquely aligns them to industrial and academic pursuits (pharmacological applications, pest control etc.) and provides a biochemical tool for studying biological activities including cell signalling and immunological response. Neogastropod snails (cones, oyster drills etc.) are carnivorous and include active predators, scavengers, grazers on sessile invertebrates and hematophagous parasites; most of them use venoms to efficiently feed. It has been hypothesized that trophic innovations were the main drivers of rapid radiation of Neogastropoda in the late Cretaceous. We present here the first molecular characterization of the alimentary secretion of a non-conoidean neogastropod, Colubraria reticulata. Colubrariids successfully feed on the blood of fishes, throughout the secretion into the host of a complex mixture of anaesthetics and anticoagulants. We used a NGS RNA-Seq approach, integrated with differential expression analyses and custom searches for putative secreted feeding-related proteins, to describe in detail the salivary and mid-oesophageal transcriptomes of this Mediterranean vampire snail, with functional and evolutionary insights on major families of bioactive molecules. RESULTS: A remarkably low level of overlap was observed between the gene expression in the two target tissues, which also contained a high percentage of putatively secreted proteins when compared to the whole body. At least 12 families of feeding-related proteins were identified, including: 1) anaesthetics, such as ShK Toxin-containing proteins and turripeptides (ion-channel blockers), Cysteine-rich secretory proteins (CRISPs), Adenosine Deaminase (ADA); 2) inhibitors of primary haemostasis, such as novel vWFA domain-containing proteins, the Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5) and the wasp Antigen-5; 3) anticoagulants, such as TFPI-like multiple Kunitz-type protease inhibitors, Peptidases S1 (PS1), CAP/ShKT domain-containing proteins, Astacin metalloproteases and Astacin/ShKT domain-containing proteins; 4) additional proteins, such the Angiotensin-Converting Enzyme (ACE: vasopressive) and the cytolytic Porins. CONCLUSIONS: Colubraria feeding physiology seems to involve inhibitors of both primary and secondary haemostasis, anaesthetics, a vasoconstrictive enzyme to reduce feeding time and tissue-degrading proteins such as Porins and Astacins. The complexity of Colubraria venomous cocktail and the divergence from the arsenal of the few neogastropods studied to date (mostly conoideans) suggest that biochemical diversification of neogastropods might be largely underestimated and worth of extensive investigation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1648-4) contains supplementary material, which is available to authorized users. |
1,773 | H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation | The host tolerance mechanisms to avian influenza virus (H5N1) infection that limit tissue injury remain unknown. Emerging evidence indicates that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(−) channel, modulates airway inflammation. Janus tyrosine kinase (JAK) 3, a JAK family member that plays a central role in inflammatory responses, prominently contributes to the dysregulated innate immune response upon H5N1 attachment; therefore, this study aims to elucidate whether JAK3 activation induced by H5N1 hemagglutinin (HA) inhibits cAMP-dependent CFTR channels. We performed short-circuit current, immunohistochemistry and molecular analyses of the airway epithelium in Jak3(+/+) and Jak3(+/−) mice. We demonstrate that H5N1 HA attachment inhibits cAMP-dependent CFTR Cl(−) channels via JAK3-mediated adenylyl cyclase (AC) suppression, which reduces cAMP production. This inhibition leads to increased nuclear factor-kappa B (NF-κB) signaling and inflammatory responses. H5N1 HA is detected by TLR4 expressed on respiratory epithelial cells, facilitating JAK3 activation. This activation induces the interaction between TLR4 and Gαi protein, which blocks ACs. Our findings provide novel insight into the pathogenesis of acute lung injury via the inhibition of cAMP-dependent CFTR channels, indicating that the administration of cAMP-elevating agents and targeting JAK3 may activate host tolerance to infection for the management of influenza virus–induced fatal pneumonia. |
1,774 | DNA transducer-triggered signal switch for visual colorimetric bioanalysis | A simple and versatile colorimetric biosensor has been developed for sensitive and specific detection of a wide range of biomolecules, such as oligonucleotides and aptamer-recognized targets. Combining the signal transducer and catalyzed hairpin assembly (CHA)-based signal amplification, the target DNA binds with the hairpin DNA to form a new nucleic acid sequence and creates a toehold in the transducer for initiating the recycle amplification reaction of CHA. The catalyzed assembly process produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimic horseradish peroxidase activity, which catalyzes a colorimetric reaction. Under optimal conditions, the calibration curve of synthetic target DNA has good linearity from 50 pM to 200 nM with a detection limit of 32 pM. This strategy has been successfully applied to detect S. pneumoniae as low as 156 CFU mL(−1), and shows a good specificity against closely related streptococci and major pathogenic bacteria. In addition, the developed method enables successful visual analysis of S. pneumoniae in clinical samples by the naked eye. Importantly, this method demonstrates excellent assay versatility for sensitively detecting oligonucleotides or aptamer-recognized targets. |
1,775 | Impact of statin therapy on mortality in patients with sepsis-associated acute respiratory distress syndrome (ARDS) depends on ARDS severity: a prospective observational cohort study | BACKGROUND: Previous investigations have presumed a potential therapeutic effect of statin therapy in patients with acute respiratory distress syndrome (ARDS). Statins are expected to attenuate inflammation in the lungs of patients with ARDS due to their anti-inflammatory effects. Clinical investigations of the role of statin therapy have revealed contradictory results. This study aimed to investigate whether pretreatment and continuous therapy with statins in patients with sepsis-associated ARDS are associated with 28-day survival according to disease severity (mild, moderate, or severe). METHODS: Patients with sepsis-associated ARDS from the surgical intensive care were enrolled in this prospective observational investigation. ARDS was classified into three groups (mild, moderate, and severe); 28-day mortality was recorded as the primary outcome variable and organ failure was recorded as secondary outcome variable. Sequential Organ Failure Assessment scores and the requirements for organ support were evaluated throughout the observational period to assess organ failure. RESULTS: 404 patients with sepsis-associated ARDS were enrolled in this investigation. The distribution of the ARDS subgroups was 13 %, 59 %, and 28 % for mild, moderate, and severe disease, respectively. Statin therapy improved 28-day survival exclusively in the patients with severe ARDS compared with patients without statin therapy (88.5 % and 62.5 %, respectively; P = 0.0193). To exclude the effects of several confounders, we performed multivariate Cox regression analysis, which showed that statin therapy remained a significant covariate for mortality (hazard ratio, 5.46; 95 % CI, 1.38–21.70; P = 0.0156). Moreover, after carrying a propensity score-matching in the severe ARDS cohort, Kaplan-Meier survival analysis confirmed the improved 28-day survival among patients with statin therapy (P = 0.0205). Patients with severe ARDS who received statin therapy had significantly more vasopressor-free days compared with those without statin therapy (13 ± 7 and 9 ± 7, respectively; P = 0.0034), and they also required less extracorporeal membrane oxygenation (ECMO) therapy and had more ECMO-free days (18 ± 9 and 15 ± 9, respectively; P = 0.0873). CONCLUSIONS: This investigation suggests a beneficial effect of continuous statin therapy in patients with severe sepsis-associated ARDS and a history of prior statin therapy. Further study is warranted to elucidate this potential effect. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-015-0368-6) contains supplementary material, which is available to authorized users. |
1,776 | Bacterial and Respiratory Viral Interactions in the Etiology of Acute Otitis Media in HIV-infected and HIV-uninfected South African Children | BACKGROUND: Bacteria and respiratory viruses are implicated in the pathogenesis of acute otitis media (AOM); however, data from low–middle income countries are sparse. We investigated the etiology of AOM in HIV-infected (HIV+), HIV-uninfected (HIV−) and HIV-exposed clinically asymptomatic for HIV-infection (HEU) South African children. METHODS: Children ≥3 months to <5 years of age with AOM were enrolled between May 2009 and April 2010 (NCT01031082). Middle ear fluid samples were cultured for bacteria; antibacterial susceptibility was done and serotyping undertaken for Streptococcus pneumoniae and Haemophilus influenzae. Nasopharyngeal aspirates were analyzed for respiratory viruses using immunofluorescence assay and polymerase chain reaction. RESULTS: Of 260 AOM episodes (HIV+:15; HIV−:182; HEU:63), bacteria were found in 54.6%, including Haemophilus influenzae (30.8%), 98.8% of which were nontypeable, and Streptococcus pneumoniae (20.4%), Staphylococcus aureus (15.8%), Moraxella catarrhalis (5.0%) and Streptococcus pyogenes (1.5%). Nonsusceptibility of Streptococcus pneumoniae to penicillin was 64.2%. Respiratory viruses were detected in 74.2% of cases. Human rhinovirus was most frequently detected (37.7%), followed by adenovirus (14.2%) and human bocavirus (11.5%) overall and irrespective of HIV status. Respiratory viruses were identified concurrently with S. pneumoniae, H. influenzae, M. catarrhalis (76.9–78.8%) and Staphylococcus aureus (63.4%) cultured from middle ear fluid, as well as in 72.0% of episodes negative for any bacteria. CONCLUSION: The study suggests that respiratory viruses and pathogenic bacteria play an important role in the development of AOM in children. A similar spectrum of pathogens was observed independently of HIV status. Vaccines targeting both nontypeable Haemophilus influenzae and S. pneumoniae may have a broad impact on AOM in South Africa. |
1,777 | 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. |
1,778 | Dietary Sodium Suppresses Digestive Efficiency via the Renin-Angiotensin System | Dietary fats and sodium are both palatable and are hypothesized to synergistically contribute to ingestive behavior and thereby obesity. Contrary to this hypothesis, C57BL/6J mice fed a 45% high fat diet exhibited weight gain that was inhibited by increased dietary sodium content. This suppressive effect of dietary sodium upon weight gain was mediated specifically through a reduction in digestive efficiency, with no effects on food intake behavior, physical activity, or resting metabolism. Replacement of circulating angiotensin II levels reversed the effects of high dietary sodium to suppress digestive efficiency. While the AT(1) receptor antagonist losartan had no effect in mice fed low sodium, the AT(2) receptor antagonist PD-123,319 suppressed digestive efficiency. Correspondingly, genetic deletion of the AT(2) receptor in FVB/NCrl mice resulted in suppressed digestive efficiency even on a standard chow diet. Together these data underscore the importance of digestive efficiency in the pathogenesis of obesity, and implicate dietary sodium, the renin-angiotensin system, and the AT(2) receptor in the control of digestive efficiency regardless of mouse strain or macronutrient composition of the diet. These findings highlight the need for greater understanding of nutrient absorption control physiology, and prompt more uniform assessment of digestive efficiency in animal studies of energy balance. |
1,779 | Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection | BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. |
1,780 | Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake | Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver. |
1,781 | Bone Regeneration Based on Tissue Engineering Conceptions — A 21st Century Perspective | The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts. |
1,782 | Pathology in Captive Wild Felids at German Zoological Gardens | This retrospective study provides an overview on spontaneous diseases occurring in 38 captive wild felids submitted for necropsy by German zoological gardens between 2004 and 2013. Species included 18 tigers, 8 leopards, 7 lions, 3 cheetahs and 2 cougars with an age ranging from 0.5 to 22 years. Renal lesions, predominantly tubular alterations (intra-tubular concrements, tubular degeneration, necrosis, intra-tubular cellular debris, proteinaceous casts, dilated tubuli) followed by interstitial (lympho-plasmacytic inflammation, fibrosis, metastatic-suppurative inflammation, eosinophilic inflammation) and glomerular lesions (glomerulonephritis, glomerulosclerosis, amyloidosis) were detected in 33 out of 38 animals (87%). Tumors were found in 19 of 38 felids (50%) with 12 animals showing more than one neoplasm. The tumor prevalence increased with age. Neoplasms originated from endocrine (11), genital (8), lympho-hematopoietic (5) and alimentary organs (4) as well as the mesothelium (3). Most common neoplasms comprised uterine/ovarian leiomyomas (5/2), thyroid adenomas/adenocarcinoma (5/1), pleural mesotheliomas (3), hemangiosarcomas (2) and glossal papillomas (2). Inflammatory changes were frequently encountered in the intestine and the lung. Two young animals displayed metastatic mineralization suggestive of a vitamin D- or calcium intoxication. One tiger exhibited degenerative white matter changes consistent with an entity termed large felid leukoencephalomyelopathy. Various hyperplastic, degenerative and inflammatory changes with minor clinical significance were found in several organs. Summarized, renal lesions followed by neoplastic changes as well as inflammatory changes in lung and gastrointestinal tract represent the most frequent findings in captive wild felids living in German zoological gardens. |
1,783 | Hepatitis C virus and host cell nuclear transport machinery: a clandestine affair | There is growing evidence that factors encoded by cytoplasmic replicating viruses functionally interact with components of the nucleocytoplasmic transport apparatus. They do so either to access the cell nucleus, thus affecting genes expression, or to interfere with nuclear transport functionality, hindering host immune response. Recent studies revealed that the hepatitis C virus (HCV) makes no exception, interacting with the host cell nuclear transport machinery at two different levels. On the one hand, small amounts of both core and NS5A localize within the host cell nucleus during productive infection, modulating gene expression and signaling functions to promote persistent infection. On the other hand, HCV infection causes a profound redistribution of certain nucleoproteins to the close proximity of endoplasmic reticulum membrane-derived viral replication factories, where viral RNA amplification occurs. These nucleoporins are believed to form nuclear pore complex-like structures, as suggested by their ability to recruit nuclear localization sequence-bearing proteins. Thus, both processes are linked to virus-induced persistence and pathogenesis, representing possible targets for the development of novel anti-HCV therapeutics. |
1,784 | BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1((-/-)) Mice from Monkeypoxvirus Lethal Challenge | Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD(106)ΔTK, BoHV-4-A-EF1α-M1RgD(106)ΔTK and BoHV-4-A-EF1α-B6RgD(106)ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD(106)ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD(106)ΔTK and BoHV-4-A-EF1α-B6RgD(106)ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1((-/-)) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform. |
1,785 | In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses | Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains. |
1,786 | Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses | Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients. |
1,787 | Timing and causes of death in septic shock | BACKGROUND: Most studies about septic shock report a crude mortality rate that neither distinguishes between early and late deaths nor addresses the direct causes of death. We herein aimed to determine the modalities of death in septic shock. METHODS: This was a 6-year (2008–2013) monocenter retrospective study. All consecutive patients diagnosed for septic shock within the first 48 h of intensive care unit (ICU) admission were included. Early and late deaths were defined as occurring within or after 3 days following ICU admission, respectively. The main cause of death in the ICU was determined from medical files. A multinomial logistic regression analysis using the status alive as the reference category was performed to identify the prognostic factors associated with early and late deaths. RESULTS: Five hundred forty-three patients were included, with a mean age of 66 ± 15 years and a high proportion (67 %) of comorbidities. The in-ICU and in-hospital mortality rates were 37.2 and 45 %, respectively. Deaths occurred early for 78 (32 %) and later on for 166 (68 %) patients in the ICU (n = 124) or in the hospital (n = 42). Early deaths were mainly attributable to intractable multiple organ failure related to the primary infection (82 %) and to mesenteric ischemia (6.4 %). In-ICU late deaths were directly related to end-of-life decisions in 29 % of patients and otherwise mostly related to ICU-acquired complications, including nosocomial infections (20.4 %) and mesenteric ischemia (16.6 %). Independent determinants of early death were age, malignancy, diabetes mellitus, no pathogen identification, and initial severity. Among 3-day survivors, independent risk factors for late death were age, cirrhosis, no pathogen identification, and previous corticosteroid treatment. CONCLUSIONS: Our study provides a comprehensive assessment of septic shock-related deaths. Identification of risk factors of early and late deaths may determine differential prognostic patterns. |
1,788 | Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis | Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies. |
1,789 | 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. |
1,790 | Successful rescue of disseminated varicella infection with multiple organ failure in a pediatric living donor liver transplant recipient: a case report and literature review | A 12-year-old female patient with biliary atresia underwent living donor liver transplantation (LDLT). Twelve months after the LDLT, she developed acute hepatitis (alanine aminotransferase 584 IU/L) and was diagnosed with disseminated varicella-zoster virus (VZV) infection with high level of serum VZV-DNA (1.5 × 10(5) copies/mL) and generalized vesicular rash. She had received the VZV vaccination when she was 5-years-old and had not been exposed to chicken pox before the LDLT, and her serum was positive for VZV immunoglobulin G at the time of the LDLT. Although she underwent treatment with intravenous acyclovir, intravenous immunoglobulin, and withdrawal of immunosuppressants, her symptoms worsened and were accompanied by disseminated intravascular coagulation, pneumonia, and encephalitis. These complications required treatment in the intensive care unit for 16 days. Five weeks later, her clinical findings improved, although her VZV-DNA levels remained high (8.5 × 10(3)copies/mL). Oral acyclovir was added for 2 weeks, and she was eventually discharged from our hospital on day 86 after admission; she has not experienced a recurrence. In conclusion, although disseminated VZV infection with multiple organ failure after pediatric LDLT is a life-threatening disease, it can be cured via an early diagnosis and intensive treatment. |
1,791 | Deep sequencing analysis of viral infection and evolution allows rapid and detailed characterization of viral mutant spectrum | Motivation: The study of RNA virus populations is a challenging task. Each population of RNA virus is composed of a collection of different, yet related genomes often referred to as mutant spectra or quasispecies. Virologists using deep sequencing technologies face major obstacles when studying virus population dynamics, both experimentally and in natural settings due to the relatively high error rates of these technologies and the lack of high performance pipelines. In order to overcome these hurdles we developed a computational pipeline, termed ViVan (Viral Variance Analysis). ViVan is a complete pipeline facilitating the identification, characterization and comparison of sequence variance in deep sequenced virus populations. Results: Applying ViVan on deep sequenced data obtained from samples that were previously characterized by more classical approaches, we uncovered novel and potentially crucial aspects of virus populations. With our experimental work, we illustrate how ViVan can be used for studies ranging from the more practical, detection of resistant mutations and effects of antiviral treatments, to the more theoretical temporal characterization of the population in evolutionary studies. Availability and implementation: Freely available on the web at http://www.vivanbioinfo.org Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online. |
1,792 | Experimental African Trypanosome Infection by Needle Passage or Natural Tsetse Fly Challenge Thwarts the Development of Collagen-Induced Arthritis in DBA/1 Prone Mice via an Impairment of Antigen Specific B Cell Autoantibody Titers | Collagen-induced arthritis is a B cell-mediated autoimmune disease. Recently published studies have demonstrated that in some rare cases pathogens can confer protection from autoimmunity. Trypanosoma brucei parasites are tsetse fly transmitted extracellular protozoans causing sleeping sickness disease in humans and Nagana in livestock in sub-Saharan endemic areas. In the past, we demonstrated that trypanosome infections impair B cell homeostasis and abolish vaccine-induced protection against unrelated antigens. Hence, here we hypothesized that trypanosome infection can affect the onset of CIA by specifically dampening specific B-cell responses and type II collagen antibody titers in DBA/1 prone mice. We observed a substantial delay in the onset of collagen-induced arthritis in T. brucei-infected DBA/1 mice that correlates with a drastic decrease of type II collagen titers of the different IgG isotypes in the serum. Treatment of infected mice with Berenil, a trypanocidal drug, restored the development of CIA-associated clinical symptoms. Interestingly, these data were confirmed by the challenge of immunized DBA/1 prone mice with T. brucei-infected tsetse flies. Together, these results demonstrate that T. brucei infection is impairing the maintenance of the antigen specific plasma B cell pool driving the development of CIA in DBA/1 prone mice. |
1,793 | Instrumental Role of Helicobacter pylori γ-Glutamyl Transpeptidase in VacA-Dependent Vacuolation in Gastric Epithelial Cells | Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases. |
1,794 | Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs | The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV. |
1,795 | Common variants of NFE2L2 gene predisposes to acute respiratory distress syndrome in patients with severe sepsis | INTRODUCTION: The purpose of this study was to investigate whether common variants across the nuclear factor erythroid 2-like 2 (NFE2L2) gene contribute to the development of the acute respiratory distress syndrome (ARDS) in patients with severe sepsis. NFE2L2 is involved in the response to oxidative stress, and it has been shown to be associated with the development of ARDS in trauma patients. METHODS: We performed a case–control study of 321 patients fulfilling international criteria for severe sepsis and ARDS who were admitted to a Spanish network of post-surgical and critical care units, as well as 871 population-based controls. Six tagging single-nucleotide polymorphisms (SNPs) of NFE2L2 were genotyped, and, after further imputation of additional 34 SNPs, association testing with ARDS susceptibility was conducted using logistic regression analysis. RESULTS: After multiple testing adjustments, our analysis revealed 10 non-coding SNPs in tight linkage disequilibrium (0.75 ≤ r(2) ≤ 1) that were associated with ARDS susceptibility as a single association signal. One of those SNPs (rs672961) was previously associated with trauma-induced ARDS and modified the promoter activity of the NFE2L2 gene, showing an odds ratio of 1.93 per T allele (95 % confidence interval, 1.17–3.18; p = 0.0089). CONCLUSIONS: Our findings support the involvement of NFE2L2 gene variants in ARDS susceptibility and reinforce further exploration of the role of oxidant stress response as a risk factor for ARDS in critically ill patients. |
1,796 | The Promise of Proteomics for the Study of ADP-Ribosylation | ADP-ribosylation is a post-translational modification where single units (mono-ADP-ribosylation) or polymeric chains (poly-ADP-ribosylation) of ADP-ribose are conjugated to proteins by ADP-ribosyltransferases. This post-translational modification and the ADP-ribosyltransferases (also known as PARPs) responsible for its synthesis have been found to play a role in nearly all major cellular processes, including DNA repair, transcription, translation, cell signaling, and cell death. Furthermore, dysregulation of ADP-ribosylation has been linked to diseases including cancers, diabetes, neurodegenerative disorders, and heart failure, leading to the development of therapeutic PARP inhibitors, many of which are currently in clinical trials. The study of this therapeutically important modification has recently been bolstered by the application of mass spectrometry-based proteomics, arguably the most powerful tool for the unbiased analysis of protein modifications. Unfortunately, progress has been hampered by the inherent challenges that stem from the physicochemical properties of ADP-ribose, which as a post-translational modification is highly charged, heterogeneous (linear or branched polymers, as well as monomers), labile, and found on a wide range of amino acid acceptors. In this Perspective, we discuss the progress that has been made in addressing these challenges, including the recent breakthroughs in proteomics techniques to identify ADP-ribosylation sites, and future developments to provide a proteome-wide view of the many cellular processes regulated by ADP-ribosylation. |
1,797 | Function-Based Mutation-Resistant Synthetic Signaling Device Activated by HIV-1 Proteolysis | [Image: see text] The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus. Many pathogens encode proteases that are essential for their replication and that have a defined substrate specificity. A genetically encoded sensor composed of a fused membrane anchor, viral protease target site, and an orthogonal transcriptional activator was engineered into a human cell line. The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes. The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors. In the future, a similar principle could be applied to detect also other pathogens and functions. |
1,798 | A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax | Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment. |
1,799 | The Use of a Shelter Software (a) to Track Frequency and Selected Risk Factors for Feline Upper Respiratory Infection | SIMPLE SUMMARY: Feline upper respiratory infection is a common disease in animal shelters. Without monitoring, effective control and prevention is difficult. We looked at a software system (a) used in shelters across the United States to determine if it can be used to track URI frequency and risk factors in a population. Reports from the software system (a) were compared to data collected manually. This showed that data currently collected were not useful for tracking URI frequency and risk factors. However, potential exists to increase the practicality and usefulness of this shelter software system to monitor URI and other diseases. ABSTRACT: Objective—Feline upper respiratory infection (URI) is a common, multi-factorial infectious disease syndrome endemic to many animal shelters. Although a significant cause of morbidity and mortality in shelter cats, URI is seldom formally monitored in shelter cat populations. Without monitoring, effective control and prevention of this often endemic disease is difficult. We looked at an integrated case management software system (a) for animal care organizations, widely used in shelters across the United States. Shelter staff routinely enter information regarding individual animals and disease status, but do not commonly use the software system to track frequency of disease. The purpose of this study was to determine if the software system (a) can be used to track URI frequency and selected risk factors in a population, and to evaluate the quality and completeness of the data as currently collected in a shelter. Design (type of study)—Descriptive Survey. Animals (or Sample)—317 cats in an animal shelter. Procedures—Reports from the software system (a) containing data regarding daily inventory, daily intake, animal identification, location, age, vaccination status, URI diagnosis and URI duration were evaluated. The reports were compared to data collected manually by an observer (Ann Therese Kommedal) to assess discrepancies, completeness, timeliness, availability and accuracy. Data were collected 6 days a week over a 4 week period. Results—Comparisons between the software system (a) reports and manually collected reports showed that 93% of inventory reports were complete and of these 99% were accurate. Fifty-two percent of the vaccination reports were complete, of which 97% were accurate. The accuracy of the software system’s age reports was 76%. Two-hundred and twenty-three cats were assigned a positive or negative URI diagnosis by the observer. The predictive value of the URI status in the software system (a) was below 60% both for positive and negative URI diagnosis. Conclusions and Clinical Relevance—data currently collected and entered into the software systems in the study shelter, was not useful for tracking URI frequency and risk factors, due to issues with both data quality and capture. However, the potential exists to increase the practicality and usefulness of this shelter software system to monitor URI and other diseases. Relevant data points, i.e., health status at intake and outcome, vaccination date and status, as well as age, should be made mandatory to facilitate more useful data collection and reporting. |
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