text: Viruses http://www.mdpi.com/journal/viruses Latest open access articles published in Viruses at http://www.mdpi.com/journal/viruses http://www.mdpi.com/1999-4915/8/10/269 Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV–host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV–host interactions, which might be more effective than the currently available anti-influenza therapeutics. Viruses 2016-09-29 8 10 Review 10.3390/v8100269 269 1999-4915 2016-09-29 doi: 10.3390/v8100269 Sandra Söderholm Yu Fu Lana Gaelings Sergey Belanov Laxman Yetukuri Mikhail Berlinkov Anton Cheltsov Simon Anders Tero Aittokallio Tuula Nyman Sampsa Matikainen Denis Kainov http://www.mdpi.com/1999-4915/8/10/267 Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. Viruses 2016-09-29 8 10 Article 10.3390/v8100267 267 1999-4915 2016-09-29 doi: 10.3390/v8100267 Mathias Schemmerer Silke Apelt Eva Trojnar Rainer Ulrich Jürgen Wenzel Reimar Johne http://www.mdpi.com/1999-4915/8/10/264 The major threat for cassava cultivation on the Indian subcontinent is cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses which are bipartite begomoviruses with DNA A and DNA B components. Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) cause CMD in India. Two isolates of SLCMV infected the cassava cultivar Sengutchi in the fields near Malappuram and Thiruvananthapuram cities of Kerala State, India. The Malappuram isolate was persistent when maintained in the Madurai Kamaraj University (MKU, Madurai, Tamil Nadu, India) greenhouse, whereas the Thiruvananthapuram isolate did not persist. The recovered cassava plants with the non-persistent SLCMV, which were maintained vegetative in quarantine in the University of Basel (Basel, Switzerland) greenhouse, displayed re-emergence of CMD after a six-month period. Interestingly, these plants did not carry SLCMV but carried ICMV. It is interpreted that the field-collected, SLCMV-infected cassava plants were co-infected with low levels of ICMV. The loss of SLCMV in recovered cassava plants, under greenhouse conditions, then facilitated the re-emergence of ICMV. The partial dimer clones of the persistent and non-persistent isolates of SLCMV and the re-emerged isolate of ICMV were infective in Nicotiana benthamiana upon agroinoculation. Studies on pseudo-recombination between SLCMV and ICMV in N. benthamiana provided evidence for trans-replication of ICMV DNA B by SLCMV DNA A. Viruses 2016-09-28 8 10 Article 10.3390/v8100264 264 1999-4915 2016-09-28 doi: 10.3390/v8100264 Chockalingam Karthikeyan Basavaprabhu Patil Basanta Borah Thulasi Resmi Silvia Turco Mikhail Pooggin Thomas Hohn Karuppannan Veluthambi http://www.mdpi.com/1999-4915/8/10/265 Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. Viruses 2016-09-28 8 10 Article 10.3390/v8100265 265 1999-4915 2016-09-28 doi: 10.3390/v8100265 Remo Leisi Marcus Von Nordheim Carlos Ros Christoph Kempf http://www.mdpi.com/1999-4915/8/10/260 Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development. Viruses 2016-09-27 8 10 Review 10.3390/v8100260 260 1999-4915 2016-09-27 doi: 10.3390/v8100260 Robert London http://www.mdpi.com/1999-4915/8/10/268 A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1–3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5′- and 3′-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1. Viruses 2016-09-27 8 10 Article 10.3390/v8100268 268 1999-4915 2016-09-27 doi: 10.3390/v8100268 Jie Zhong Xi Pang Hong Zhu Bi Gao Wen Huang Qian Zhou http://www.mdpi.com/1999-4915/8/10/266 Herpesviruses use a vesicle-mediated transfer of intranuclearly assembled nucleocapsids through the nuclear envelope (NE) for final maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex (NEC), conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane (INM) by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane (ONM). Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the NE has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport. Viruses 2016-09-27 8 10 Commentary 10.3390/v8100266 266 1999-4915 2016-09-27 doi: 10.3390/v8100266 Thomas Mettenleiter http://www.mdpi.com/1999-4915/8/10/263 Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromatic π–π stacking plays a crucial role in the binding of NNRTIs to RT. Viruses 2016-09-23 8 10 Article 10.3390/v8100263 263 1999-4915 2016-09-23 doi: 10.3390/v8100263 Ming-Tain Lai Vandna Munshi Meiqing Lu MeiZhen Feng Renee Hrin-Solt Philip McKenna Daria Hazuda Michael Miller http://www.mdpi.com/1999-4915/8/9/258 Capsid-targeted viral inactivation (CTVI), a conceptually powerful new antiviral strategy, is attracting increasing attention from researchers. Specifically, this strategy is based on fusion between the capsid protein of a virus and a crucial effector molecule, such as a nuclease (e.g., staphylococcal nuclease, Barrase, RNase HI), lipase, protease, or single-chain antibody (scAb). In general, capsid proteins have a major role in viral integration and assembly, and the effector molecule used in CTVI functions to degrade viral DNA/RNA or interfere with proper folding of viral key proteins, thereby affecting the infectivity of progeny viruses. Interestingly, such a capsid–enzyme fusion protein is incorporated into virions during packaging. CTVI is more efficient compared to other antiviral methods, and this approach is promising for antiviral prophylaxis and therapy. This review summarizes the mechanism and utility of CTVI and provides some successful applications of this strategy, with the ultimate goal of widely implementing CTVI in antiviral research. Viruses 2016-09-21 8 9 Review 10.3390/v8090258 258 1999-4915 2016-09-21 doi: 10.3390/v8090258 Xingcui Zhang Renyong Jia Jiakun Zhou Mingshu Wang Zhongqiong Yin Anchun Cheng http://www.mdpi.com/1999-4915/8/9/262 Alphaherpesviruses are highly prevalent in equine populations and co-infections with more than one of these viruses' strains frequently diagnosed. Lytic replication and latency with subsequent reactivation, along with new episodes of disease, can be influenced by genetic diversity generated by spontaneous mutation and recombination. Latency enhances virus survival by providing an epidemiological strategy for long-term maintenance of divergent strains in animal populations. The alphaherpesviruses equine herpesvirus 1 (EHV-1) and 9 (EHV-9) have recently been shown to cross species barriers, including a recombinant EHV-1 observed in fatal infections of a polar bear and Asian rhinoceros. Little is known about the latency and genetic diversity of EHV-1 and EHV-9, especially among zoo and wild equids. Here, we report evidence of limited genetic diversity in EHV-9 in zebras, whereas there is substantial genetic variability in EHV-1. We demonstrate that zebras can be lytically and latently infected with both viruses concurrently. Such a co-occurrence of infection in zebras suggests that even relatively slow-evolving viruses such as equine herpesviruses have the potential to diversify rapidly by recombination. This has potential consequences for the diagnosis of these viruses and their management in wild and captive equid populations. Viruses 2016-09-20 8 9 Article 10.3390/v8090262 262 1999-4915 2016-09-20 doi: 10.3390/v8090262 Azza Abdelgawad Armando Damiani Simon Ho Günter Strauss Claudia Szentiks Marion East Nikolaus Osterrieder Alex Greenwood http://www.mdpi.com/1999-4915/8/9/253 Hepatitis E virus (HEV), an RNA virus of the Hepeviridae family, has marked heterogeneity. While all five HEV genotypes can cause human infections, genotypes HEV-1 and -2 infect humans alone, genotypes HEV-3 and -4 primarily infect pigs, boars and deer, and genotype HEV-7 primarily infects dromedaries. The global distribution of HEV has distinct epidemiological patterns based on ecology and socioeconomic factors. In resource-poor countries, disease presents as large-scale waterborne epidemics, and few epidemics have spread through person-to-person contact; however, endemic diseases within these countries can potentially spread through person-to-person contact or fecally contaminated water and foods. Vertical transmission of HEV from infected mother to fetus causes high fetal and perinatal mortality. Other means of transmission, such as zoonotic transmission, can fluctuate depending upon the region and strain of the virus. For instance, zoonotic transmission can sometimes play an insignificant role in human infections, such as in India, where human and pig HEV infections are unrelated. However, recently China and Southeast Asia have experienced a zoonotic spread of HEV-4 from pigs to humans and this has become the dominant mode of transmission of hepatitis E in eastern China. Zoonotic HEV infections in humans occur by eating undercooked pig flesh, raw liver, and sausages; through vocational contact; or via pig slurry, which leads to environmental contamination of agricultural products and seafood. Lastly, blood transfusion-associated HEV infections occur in many countries and screening of donors for HEV RNA is currently under serious consideration. To summarize, HEV genotypes 1 and 2 cause epidemic and endemic diseases in resource poor countries, primarily spreading through contaminated drinking water. HEV genotypes 3 and 4 on the other hand, cause autochthonous infections in developed, and many developing countries, by means of a unique zoonotic food-borne transmission. Viruses 2016-09-20 8 9 Review 10.3390/v8090253 253 1999-4915 2016-09-20 doi: 10.3390/v8090253 Mohammad Khuroo Mehnaaz Khuroo Naira Khuroo http://www.mdpi.com/1999-4915/8/9/261 Chronic hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are major causes of liver disease and hepatocellular carcinoma worldwide. Despite the presence of an efficient preventive vaccine, more than 250 million patients are chronically infected with HBV. Current antivirals effectively control but only rarely cure chronic infection. While the molecular biology of the two viruses has been characterized in great detail, the absence of robust cell culture models for HBV and/or HDV infection has limited the investigation of virus-host interactions. Native hepatoma cell lines do not allow viral infection, and the culture of primary hepatocytes, the natural host cell for the viruses, implies a series of constraints restricting the possibilities of analyzing virus-host interactions. Recently, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key HBV/HDV cell entry factor has opened the door to a new era of investigation, as NTCP-overexpressing hepatoma cells acquire susceptibility to HBV and HDV infections. In this review, we summarize the major cell culture models for HBV and HDV infection, discuss their advantages and limitations and highlight perspectives for future developments. Viruses 2016-09-20 8 9 Review 10.3390/v8090261 261 1999-4915 2016-09-20 doi: 10.3390/v8090261 Eloi Verrier Che Colpitts Catherine Schuster Mirjam Zeisel Thomas Baumert http://www.mdpi.com/1999-4915/8/9/257 Infectious retrovirus particles contain two copies of unspliced viral RNA that serve as the viral genome. Unspliced retroviral RNA is transcribed in the nucleus by the host RNA polymerase II and has three potential fates: (1) it can be spliced into subgenomic messenger RNAs (mRNAs) for the translation of viral proteins; or it can remain unspliced to serve as either (2) the mRNA for the translation of Gag and Gag–Pol; or (3) the genomic RNA (gRNA) that is packaged into virions. The Gag structural protein recognizes and binds the unspliced viral RNA to select it as a genome, which is selected in preference to spliced viral RNAs and cellular RNAs. In this review, we summarize the current state of understanding about how retroviral packaging is orchestrated within the cell and explore potential new mechanisms based on recent discoveries in the field. We discuss the cis-acting elements in the unspliced viral RNA and the properties of the Gag protein that are required for their interaction. In addition, we discuss the role of host factors in influencing the fate of the newly transcribed viral RNA, current models for how retroviruses distinguish unspliced viral mRNA from viral genomic RNA, and the possible subcellular sites of genomic RNA dimerization and selection by Gag. Although this review centers primarily on the wealth of data available for the alpharetrovirus Rous sarcoma virus, in which a discrete RNA packaging sequence has been identified, we have also summarized the cis- and trans-acting factors as well as the mechanisms governing gRNA packaging of other retroviruses for comparison. Viruses 2016-09-20 8 9 Review 10.3390/v8090257 257 1999-4915 2016-09-20 doi: 10.3390/v8090257 Rebecca Kaddis Maldonado Leslie Parent http://www.mdpi.com/1999-4915/8/9/250 The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles "blind" to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces. Viruses 2016-09-20 8 9 Review 10.3390/v8090250 250 1999-4915 2016-09-20 doi: 10.3390/v8090250 Liang-Tzung Lin Christopher Richardson http://www.mdpi.com/1999-4915/8/9/259 Hepatitis E virus (HEV) is a human pathogen with zoonotic spread, infecting both domestic and wild animals. About 17% of the Swedish population is immune to HEV, but few cases are reported annually, indicating that most infections are subclinical. However, clinical hepatitis E may also be overlooked. For identified cases, the source of infection is mostly unknown. In order to identify whether HEV may be spread from wild game, the prevalence of markers for past and/or ongoing infection was investigated in sera and stool samples collected from 260 hunted Swedish wild ungulates. HEV markers were found in 43 (17%) of the animals. The most commonly infected animal was moose (Alces alces) with 19 out of 69 animals (28%) showing HEV markers, followed by wild boar (Sus scrofa) with 21 out of 139 animals (15%), roe deer (Capreolus capreolus) with 2 out of 30 animals, red deer (Cervus elaphus) with 1 out of 15 animals, and fallow deer (Dama dama) 0 out of 7 animals. Partial open reading frame 1 (ORF1) of the viral genomes from the animals were sequenced and compared with those from 14 endemic human cases. Phylogenetic analysis revealed that three humans were infected with HEV strains similar to those from wild boar. These results indicate that wild animals may be a source of transmission to humans and could be an unrecognized public health concern. Viruses 2016-09-19 8 9 Article 10.3390/v8090259 259 1999-4915 2016-09-19 doi: 10.3390/v8090259 Anette Roth Jay Lin Lars Magnius Marie Karlsson Sándór Belák Frederik Widén Heléne Norder http://www.mdpi.com/1999-4915/8/9/256 Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The "psi" (Ψ) element within the 5′-untranslated region (5′UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity. Viruses 2016-09-19 8 9 Article 10.3390/v8090256 256 1999-4915 2016-09-19 doi: 10.3390/v8090256 Tiffiny Rye-McCurdy Erik Olson Shuohui Liu Christiana Binkley Joshua-Paolo Reyes Brian Thompson John Flanagan Leslie Parent Karin Musier-Forsyth http://www.mdpi.com/1999-4915/8/9/255 Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed to potentiate or inhibit viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells. Viruses 2016-09-19 8 9 Review 10.3390/v8090255 255 1999-4915 2016-09-19 doi: 10.3390/v8090255 Dylan Frabutt Yong-Hui Zheng http://www.mdpi.com/1999-4915/8/9/254 Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses. Viruses 2016-09-16 8 9 Article 10.3390/v8090254 254 1999-4915 2016-09-16 doi: 10.3390/v8090254 Sarah Moeschler Samira Locher Karl-Klaus Conzelmann Beate Krämer Gert Zimmer http://www.mdpi.com/1999-4915/8/9/247 This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses. Viruses 2016-09-13 8 9 Article 10.3390/v8090247 247 1999-4915 2016-09-13 doi: 10.3390/v8090247 Alice Labaronne Christopher Swale Alexandre Monod Guy Schoehn Thibaut Crépin Rob Ruigrok http://www.mdpi.com/1999-4915/8/9/246 Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag. Viruses 2016-09-12 8 9 Review 10.3390/v8090246 246 1999-4915 2016-09-12 doi: 10.3390/v8090246 Mauricio Comas-Garcia Sean Davis Alan Rein http://www.mdpi.com/1999-4915/8/9/251 The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pave the path towards the structure-based design and optimization of next-generation RdRp inhibitors. Viruses 2016-09-10 8 9 Review 10.3390/v8090251 251 1999-4915 2016-09-10 doi: 10.3390/v8090251 Julien Sourimant Richard Plemper http://www.mdpi.com/1999-4915/8/9/248 Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles. Viruses 2016-09-10 8 9 Review 10.3390/v8090248 248 1999-4915 2016-09-10 doi: 10.3390/v8090248 Elodie Mailler Serena Bernacchi Roland Marquet Jean-Christophe Paillart Valérie Vivet-Boudou Redmond Smyth http://www.mdpi.com/1999-4915/8/9/249 The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft. Viruses 2016-09-10 8 9 Article 10.3390/v8090249 249 1999-4915 2016-09-10 doi: 10.3390/v8090249 Naoki Takizawa Fumitaka Momose Yuko Morikawa Akio Nomoto http://www.mdpi.com/1999-4915/8/9/252 Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells. Viruses 2016-09-10 8 9 Communication 10.3390/v8090252 252 1999-4915 2016-09-10 doi: 10.3390/v8090252 Luwanika Mlera Wessam Melik Danielle Offerdahl Eric Dahlstrom Stephen Porcella Marshall Bloom http://www.mdpi.com/1999-4915/8/9/245 The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV−ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck®) and cytology. This showed 22/113 (19.5%) of LBC's from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV−ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07–26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV−ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear. Viruses 2016-09-05 8 9 Article 10.3390/v8090245 245 1999-4915 2016-09-05 doi: 10.3390/v8090245 Xiaotong He Innocent Maranga Anthony Oliver Peter Gichangi Lynne Hampson Ian Hampson http://www.mdpi.com/1999-4915/8/9/244 Both temperature and humidity may independently or jointly contribute to the risk of human rhinovirus (HRV) infections, either through altered survival and spread of viruses in the environment or due to changes in host susceptibility. This study examined the relationship between short-term variations in temperature and humidity and the risk of HRV infections in a subarctic climate. We conducted a case-crossover study among conscripts (n = 892) seeking medical attention due to respiratory symptoms during their military training and identified 147 HRV cases by real-time PCR. An average temperature, a decline in daily ambient temperature and absolute humidity (AH) during the three preceding days of the onset (hazard period) and two reference periods (a week prior and after the onset) were obtained. The average daily temperature preceding HRV infections was −9.9 ± 4.9 °C and the average AH was 2.2 ± 0.9 g/m3. An average (odds ratios (OR) 1.07 (95% confidence interval (CI) 1.00–1.15)) and maximal (OR 1.08 (1.01–1.17)) change in temperature increased the risk of HRV infections by 8% per 1 °C decrease. An average (OR 1.20 (CI 1.03–1.40)) and maximal decrease (OR 1.13 (CI 0.96–1.34)) in AH increased the risk of HRV infection by 13% and 20% per 0.5 g/m3 decrease. A higher average temperature during the three preceding days was positively associated with HRV infections (OR 1.07 (CI 1.00–1.15)). A decrease rather than low temperature and humidity per se during the preceding few days increases the risk of HRV infections in a cold climate. The information is applicable to populations residing in cold climates for appropriate personal protection and prevention of adverse health effects. Viruses 2016-09-02 8 9 Article 10.3390/v8090244 244 1999-4915 2016-09-02 doi: 10.3390/v8090244 Tiina Ikäheimo Kari Jaakkola Jari Jokelainen Annika Saukkoriipi Merja Roivainen Raija Juvonen Olli Vainio Jouni Jaakkola http://www.mdpi.com/1999-4915/8/9/241 The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions. Viruses 2016-08-30 8 9 Article 10.3390/v8090241 241 1999-4915 2016-08-30 doi: 10.3390/v8090241 Yung Lee Sara Dizzell Vivian Leung Aisha Nazli Muhammad Zahoor Raina Fichorova Charu Kaushic http://www.mdpi.com/1999-4915/8/9/243 Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. Viruses 2016-08-30 8 9 Review 10.3390/v8090243 243 1999-4915 2016-08-30 doi: 10.3390/v8090243 Martin Hamann Dirk Lindemann http://www.mdpi.com/1999-4915/8/9/242 To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases. Viruses 2016-08-29 8 9 Review 10.3390/v8090242 242 1999-4915 2016-08-29 doi: 10.3390/v8090242 Allison Dupzyk Billy Tsai http://www.mdpi.com/1999-4915/8/8/240 One of the major hurdles to porcine reproductive and respiratory syndrome (PRRS) vaccinology is the limited or no cross-protection conferred by current vaccines. To overcome this challenge, a PRRS chimeric virus (CV) was constructed using an FL12-based cDNA infectious clone in which open reading frames (ORFs) 3–4 and ORFs 5–6 were replaced with the two Korean field isolates K08-1054 and K07-2273,respectively. This virus was evaluated as a vaccine candidate to provide simultaneous protection against two genetically distinct PRRS virus (PRRSV) strains. Thirty PRRS-negative three-week-old pigs were divided into five groups and vaccinated with CV, K08-1054, K07-2273, VR-2332, or a mock inoculum. At 25 days post-vaccination (dpv), the pigs in each group were divided further into two groups and challenged with either K08-1054 or K07-2273. All of the pigs were observed until 42 dpv and were euthanized for pathological evaluation. Overall, the CV-vaccinated group exhibited higher levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-12 (IL-12) expression and of serum virus-neutralizing antibodies compared with the other groups after vaccination and also demonstrated better protection levels against both viruses compared with the challenge control group. Based on these results, it was concluded that CV might be an effective vaccine model that can confer a broader range of cross-protection to various PRRSV strains. Viruses 2016-08-22 8 8 Article 10.3390/v8080240 240 1999-4915 2016-08-22 doi: 10.3390/v8080240 Nadeem Shabir Amina Khatun Salik Nazki Bumseok Kim Eun-Jin Choi Dong Sun Kyoung-Jin Yoon Won-Il Kim http://www.mdpi.com/1999-4915/8/8/232 Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes. Viruses 2016-08-22 8 8 Article 10.3390/v8080232 232 1999-4915 2016-08-22 doi: 10.3390/v8080232 Tanja Vollmer Juergen Diekmann Matthias Eberhardt Cornelius Knabbe Jens Dreier http://www.mdpi.com/1999-4915/8/8/237 Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. Viruses 2016-08-22 8 8 Article 10.3390/v8080237 237 1999-4915 2016-08-22 doi: 10.3390/v8080237 Claire Smith Paul Scott Christopher O'Callaghan Andrew Easton Nigel Dimmock http://www.mdpi.com/1999-4915/8/8/239 In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. Viruses 2016-08-22 8 8 Review 10.3390/v8080239 239 1999-4915 2016-08-22 doi: 10.3390/v8080239 Mireia Ferrer Simon Henriet Célia Chamontin Sébastien Lainé Marylène Mougel http://www.mdpi.com/1999-4915/8/8/238 The influenza A virus genome comprises eight negative-sense viral RNAs (vRNAs) that form individual ribonucleoprotein (RNP) complexes. In order to incorporate a complete set of each of these vRNAs, the virus uses a selective packaging mechanism that facilitates co-packaging of specific gene segments but whose molecular basis is still not fully understood. Recently, we used a competitive transfection model where plasmids encoding the A/Puerto Rico/8/34 (PR8) and A/Udorn/307/72 (Udorn) PB1 gene segments were competed to show that the Udorn PB1 gene segment is preferentially co-packaged into progeny virions with the Udorn NA gene segment. Here we created chimeric PB1 genes combining both Udorn and PR8 PB1 sequences to further define the location within the Udorn PB1 gene that drives co-segregation of these genes and show that nucleotides 1776–2070 of the PB1 gene are crucial for preferential selection. In vitro assays examining specific interactions between Udorn NA vRNA and purified vRNAs transcribed from chimeric PB1 genes also supported the importance of this region in the PB1-NA interaction. Hence, this work identifies an association between viral genes that are co-selected during packaging. It also reveals a region potentially important in the RNP-RNP interactions within the supramolecular complex that is predicted to form prior to budding to allow one of each segment to be packaged in the viral progeny. Our study lays the foundation to understand the co-selection of specific genes, which may be critical to the emergence of new viruses with pandemic potential. Viruses 2016-08-20 8 8 Article 10.3390/v8080238 238 1999-4915 2016-08-20 doi: 10.3390/v8080238 Brad Gilbertson Tian Zheng Marie Gerber Anne Printz-Schweigert Chi Ong Roland Marquet Catherine Isel Steven Rockman Lorena Brown http://www.mdpi.com/1999-4915/8/8/236 In Corsica, extensive pig breeding systems allow frequent interactions between wild boars and domestic pigs, which are suspected to act as reservoirs of several zoonotic diseases including hepatitis E virus (HEV). In this context, 370 sera and 166 liver samples were collected from phenotypically characterized as pure or hybrid wild boars, between 2009 and 2012. In addition, serum and liver from 208 domestic pigs belonging to 30 farms were collected at the abattoir during the end of 2013. Anti-HEV antibodies were detected in 26% (21%–31.6%) of the pure wild boar, 43.5% (31%–56.7%) of hybrid wild boar and 88% (82.6%–91.9%) of the domestic pig sera. In addition, HEV RNA was detected in five wild boars, three hybrid wild boars and two domestic pig livers tested. Our findings provide evidence that both domestic pig and wild boar (pure and hybrid) act as reservoirs of HEV in Corsica, representing an important zoonotic risk for Corsican hunters and farmers but also for the large population of consumers of raw pig liver specialties produced in Corsica. In addition, hybrid wild boars seem to play an important ecological role in the dissemination of HEV between domestic pig and wild boar populations, unnoticed to date, that deserves further investigation. Viruses 2016-08-20 8 8 Article 10.3390/v8080236 236 1999-4915 2016-08-20 doi: 10.3390/v8080236 Ferran Jori Morgane Laval Oscar Maestrini François Casabianca François Charrier Nicole Pavio http://www.mdpi.com/1999-4915/8/8/234 The objective of this study is primarily to compare the performance of the VIDAS® Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost® Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA® (Microimmune). The sensitivity and the agreement of the VIDAS® Measles IgG assay compared to the Enzygnost® Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA® assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS® Measles IgG assay to measure anti-measles virus IgG antibody avidity with the help of the VIDAS® CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI) < 0.3 and a strong avidity as an AI > 0.6. The VIDAS® Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects. Viruses 2016-08-20 8 8 Article 10.3390/v8080234 234 1999-4915 2016-08-20 doi: 10.3390/v8080234 Julia Dina Christian Creveuil Stephanie Gouarin Florent Viron Amelie Hebert Francois Freymuth Astrid Vabret http://www.mdpi.com/1999-4915/8/8/235 As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs' packaging determinants differ from the viral genome's, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive. Viruses 2016-08-19 8 8 Review 10.3390/v8080235 235 1999-4915 2016-08-19 doi: 10.3390/v8080235 Alice Telesnitsky Sandra Wolin http://www.mdpi.com/1999-4915/8/8/233 The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. Viruses 2016-08-19 8 8 Article 10.3390/v8080233 233 1999-4915 2016-08-19 doi: 10.3390/v8080233 Florence Nicot Michelle Cazabat Sébastien Lhomme Olivier Marion Karine Sauné Julie Chiabrando Martine Dubois Nassim Kamar Florence Abravanel Jacques Izopet http://www.mdpi.com/1999-4915/8/8/231 Rabies has affected mankind for several centuries and is one of the oldest known zoonoses. It is peculiar how little is known regarding the means by which rabies virus (RABV) evades the immune response and kills its host. This review investigates the complex interplay between RABV and the immune system, including the various means by which RABV evades, or advantageously utilizes, the host immune response in order to ensure successful replication and spread to another host. Different factors that influence immune responses—including age, sex, cerebral lateralization and temperature—are discussed, with specific reference to RABV and the effects on host morbidity and mortality. We also investigate the role of apoptosis and discuss whether it is a detrimental or beneficial mechanism of the host's response to infection. The various RABV proteins and their roles in immune evasion are examined in depth with reference to important domains and the downstream effects of these interactions. Lastly, an overview of the means by which RABV evades important immune responses is provided. The research discussed in this review will be important in determining the roles of the immune response during RABV infections as well as to highlight important therapeutic target regions and potential strategies for rabies treatment. Viruses 2016-08-19 8 8 Review 10.3390/v8080231 231 1999-4915 2016-08-19 doi: 10.3390/v8080231 Terence Scott Louis Nel http://www.mdpi.com/1999-4915/8/8/230 The Middle East respiratory syndrome coronavirus (MERS-CoV) was first recognized in 2012 and can cause severe disease in infected humans. Dromedary camels are the reservoir for the virus, although, other than nasal discharge, these animals do not display any overt clinical disease. Data from in vitro experiments suggest that other livestock such as sheep, goats, and horses might also contribute to viral transmission, although field data has not identified any seropositive animals. In order to understand if these animals could be infected, we challenged young goats and horses and adult sheep with MERS-CoV by intranasal inoculation. Minimal or no virus shedding was detected in all of the animals. During the four weeks following inoculation, neutralizing antibodies were detected in the young goats, but not in sheep or horses. Viruses 2016-08-19 8 8 Article 10.3390/v8080230 230 1999-4915 2016-08-19 doi: 10.3390/v8080230 Danielle Adney Vienna Brown Stephanie Porter Helle Bielefeldt-Ohmann Airn Hartwig Richard Bowen http://www.mdpi.com/1999-4915/8/8/227 Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. Viruses 2016-08-18 8 8 Article 10.3390/v8080227 227 1999-4915 2016-08-18 doi: 10.3390/v8080227 Yonglin Yang Shaoli Lin Yuchen Nan Zexu Ma Liping Yang Yanjin Zhang http://www.mdpi.com/1999-4915/8/8/229 Hepatitis E virus (HEV), an enterically transmitted hepatotropic virus, was thought to be non-enveloped for decades. However, recent studies have revealed that the virus circulating in the patient's blood is completely cloaked in host membranes and resistant to neutralizing antibodies. The discovery of this novel enveloped form of HEV has raised a series of questions about the fundamental biology of HEV and the way this virus, which has been understudied in the past, interacts with its host. Here, we review recent advances towards understanding this phenomenon and discuss its potential impact on various aspects of the HEV life cycle and immunity. Viruses 2016-08-18 8 8 Review 10.3390/v8080229 229 1999-4915 2016-08-18 doi: 10.3390/v8080229 Xin Yin Xinlei Li Zongdi Feng http://www.mdpi.com/1999-4915/8/8/219 The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. Viruses 2016-08-18 8 8 Article 10.3390/v8080219 219 1999-4915 2016-08-18 doi: 10.3390/v8080219 Mirjam Steingruber Alexandra Kraut Eileen Socher Heinrich Sticht Anna Reichel Thomas Stamminger Bushra Amin Yohann Couté Corina Hutterer Manfred Marschall http://www.mdpi.com/1999-4915/8/8/228 Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the "life aspects" of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant. Viruses 2016-08-18 8 8 Review 10.3390/v8080228 228 1999-4915 2016-08-18 doi: 10.3390/v8080228 Mattia Dall'Ara Claudio Ratti Salah Bouzoubaa David Gilmer http://www.mdpi.com/1999-4915/8/8/226 Chronic hepatitis C is a major cause of chronic liver disease, including liver cirrhosis and hepatocellular carcinoma. The development of direct-acting antivirals (DAAs) revolutionized hepatitis C virus (HCV) treatment by offering genuine prospects for the first comprehensive cure of a chronic viral infection in humans. While antiviral resistance is a significant limitation for interferon-based therapies, resistance and treatment failure still appear to be present in a small fraction of patients even in state-of-the-art DAA combination therapies. Therefore, treatment failure and resistance still remain a clinical challenge for the management of patients not responding to DAAs. In this special issue of Viruses on HCV drug resistance, mechanisms of antiviral resistance for different classes of antiviral drugs are described. Furthermore, the detection and monitoring of resistance in clinical practice, the clinical impact of resistance in different patient groups and strategies to prevent and address resistance and treatment failure using complementary antiviral strategies are reviewed. Viruses 2016-08-16 8 8 Editorial 10.3390/v8080226 226 1999-4915 2016-08-16 doi: 10.3390/v8080226 Che Colpitts Thomas Baumert http://www.mdpi.com/1999-4915/8/8/222 Hepatitis E virus (HEV) infection can cause hepatic and extra-hepatic manifestations. Treatment of HEV infection has been thoroughly studied in solid-organ-transplant patients who have developed a chronic HEV infection. In this review, we report on our current knowledge regarding treatment of HEV infection. Viruses 2016-08-15 8 8 Review 10.3390/v8080222 222 1999-4915 2016-08-15 doi: 10.3390/v8080222 Nassim Kamar Sébastien Lhomme Florence Abravanel Olivier Marion Jean-Marie Peron Laurent Alric Jacques Izopet http://www.mdpi.com/1999-4915/8/8/220 Powassan virus (POWV) belongs to the family Flaviviridae and is a member of the tick-borne encephalitis serogroup. Transmission of POWV from infected ticks to humans has been documented in the USA, Canada, and Russia, causing fatal encephalitis in 10% of human cases and significant neurological sequelae in survivors. We used C57BL/6 mice to investigate POWV infection and pathogenesis. After footpad inoculation, infected animals exhibited rapid disease progression and 100% mortality. Immunohistochemistry and immunofluorescence revealed a very strong neuronal tropism of POWV infection. The central nervous system infection appeared as a meningoencephalitis with perivascular mononuclear infiltration and microglial activation in the brain, and a poliomyelitis-like syndrome with high level of POWV antigen at the ventral horn of the spinal cord. Pathological studies also revealed substantial infection of splenic macrophages by POWV, which suggests that the spleen plays a more important role in pathogenesis than previously realized. This report provides a detailed description of the neuroanatomical distribution of the lesions produced by POWV infection in C57BL/6 mice. Viruses 2016-08-12 8 8 Article 10.3390/v8080220 220 1999-4915 2016-08-12 doi: 10.3390/v8080220 Rodrigo Santos Meghan Hermance Benjamin Gelman Saravanan Thangamani http://www.mdpi.com/1999-4915/8/8/223 Hantaviruses, like other members of the Bunyaviridae family, are emerging viruses that are able to cause hemorrhagic fevers. Occasional transmission to humans is due to inhalation of contaminated aerosolized excreta from infected rodents. Hantaviruses are asymptomatic in their rodent or insectivore natural hosts with which they have co-evolved for millions of years. In contrast, hantaviruses cause different pathologies in humans with varying mortality rates, depending on the hantavirus species and its geographic origin. Cases of hemorrhagic fever with renal syndrome (HFRS) have been reported in Europe and Asia, while hantavirus cardiopulmonary syndromes (HCPS) are observed in the Americas. In some cases, diseases caused by Old World hantaviruses exhibit HCPS-like symptoms. Although the etiologic agents of HFRS were identified in the early 1980s, the way hantaviruses interact with their different hosts still remains elusive. What are the entry receptors? How do hantaviruses propagate in the organism and how do they cope with the immune system? This review summarizes recent data documenting interactions established by pathogenic and nonpathogenic hantaviruses with their natural or human hosts that could highlight their different outcomes. Viruses 2016-08-11 8 8 Review 10.3390/v8080223 223 1999-4915 2016-08-11 doi: 10.3390/v8080223 Myriam Ermonval Florence Baychelier Noël Tordo http://www.mdpi.com/1999-4915/8/8/225 Virus infection frequently modifies plant phenotypes, leading to changes in behaviour and performance of their insect vectors in a way that transmission is enhanced, although this may not always be the case. Here, we investigated Bemisia tabaci response to tomato plants infected by Tomato chlorosis virus (ToCV), a non-circulative-transmitted crinivirus, and Tomato severe rugose virus (ToSRV), a circulative-transmitted begomovirus. Moreover, we examined the role of visual and olfactory cues in host plant selection by both viruliferous and non-viruliferous B. tabaci. Visual cues alone were assessed as targets for whitefly landing by placing leaves underneath a Plexiglas plate. A dual-choice arena was used to assess whitefly response to virus-infected and mock-inoculated tomato leaves under light and dark conditions. Thereafter, we tested the whitefly response to volatiles using an active air-flow Y-tube olfactometer, and chemically characterized the blends using gas chromatography coupled to mass spectrometry. Visual stimuli tests showed that whiteflies, irrespective of their infectious status, always preferred to land on virus-infected rather than on mock-inoculated leaves. Furthermore, whiteflies had no preference for either virus-infected or mock-inoculated leaves under dark conditions, but preferred virus-infected leaves in the presence of light. ToSRV-infection promoted a sharp decline in the concentration of some tomato volatiles, while an increase in the emission of some terpenes after ToCV infection was found. ToSRV-viruliferous whiteflies preferred volatiles emitted from mock-inoculated plants, a conducive behaviour to enhance virus spread, while volatiles from ToCV-infected plants were avoided by non-viruliferous whiteflies, a behaviour that is likely detrimental to the secondary spread of the virus. In conclusion, the circulative persistent begomovirus, ToSRV, seems to have evolved together with its vector B. tabaci to optimise its own spread. However, this type of virus-induced manipulation of vector behaviour was not observed for the semi persistent crinivirus, ToCV, which is not specifically transmitted by B. tabaci and has a much less intimate virus-vector relationship. Viruses 2016-08-11 8 8 Article 10.3390/v8080225 225 1999-4915 2016-08-11 doi: 10.3390/v8080225 Alberto Fereres Maria Peñaflor Carla Favaro Kamila Azevedo Carolina Landi Nathalie Maluta José Bento Joao Lopes http://www.mdpi.com/1999-4915/8/8/221 Short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that analysis of telomere length (TL) in human T-cell lymphotropic virus (HTLV) infection and HTLV-associated neuropathy might provide clues to the etiology of HTLV-associated disease and viral dynamics. A subset of 45 human T-cell lymphotropic virus type 1 (HTLV-1), 45 human T-cell lymphotropic virus type 2 (HTLV-2), and 45 seronegative subjects was selected from the larger HTLV Outcomes Study (HOST) cohort, matched on age, sex and race/ethnicity. Telomere-to-single-copy gene (T/S) ratio (a measure of TL) and HTLV-1 and HTLV-2 proviral loads were measured in peripheral blood mononuclear cells (PBMCs) using quantitative PCR (qPCR). Vibration sensation measured by tuning fork during neurologic examinations performed as part of the HOST study allowed for an assessment of peripheral neuropathy. TL was compared between groups using t-tests, linear and logistic regression. Mean T/S ratio was 1.02 ± 0.16 in HTLV-1, 1.03 ± 0.17 in HTLV-2 and 0.99 ± 0.18 in HTLV seronegative subjects (p = 0.322). TL was not associated with HTLV-1 or -2 proviral load. Shorter TL was significantly associated with impaired vibration sense in the HTLV-2 positive group only. Overall, we found no evidence that telomere length was affected by chronic HTLV-1 and HTLV-2 infection. That TL was only associated with peripheral neuropathy in the HTLV-2-positive group is intriguing, but should be interpreted cautiously. Studies with larger sample size and telomere length measurement in lymphocyte subsets may clarify the relationship between TL and HTLV-infection. Viruses 2016-08-11 8 8 Article 10.3390/v8080221 221 1999-4915 2016-08-11 doi: 10.3390/v8080221 Benjamin Usadi Roberta Bruhn Jue Lin Tzong-Hae Lee Elizabeth Blackburn Edward Murphy http://www.mdpi.com/1999-4915/8/8/224 In the last 25 years, the scientific and public attention paid to bunyaviruses has increased considerably.[...] Viruses 2016-08-10 8 8 Obituary 10.3390/v8080224 224 1999-4915 2016-08-10 doi: 10.3390/v8080224 Alain Kohl Benjamin Brennan Friedemann Weber http://www.mdpi.com/1999-4915/8/8/218 The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level. Viruses 2016-08-09 8 8 Review 10.3390/v8080218 218 1999-4915 2016-08-09 doi: 10.3390/v8080218 Catherine Isel Sandie Munier Nadia Naffakh http://www.mdpi.com/1999-4915/8/8/211 There have been large numbers of studies on anti-HEV IgG seroprevalence in Europe, however, the results of these studies have produced high variability of seroprevalence rates, making interpretation increasingly problematic. Therefore, the aim of this study was to develop a clearer understanding of anti-HEV IgG seroprevalence in Europe and identify risk groups for HEV exposure by a meta-analysis of published studies. Methods: All European HEV-seroprevalence studies from 2003 to 2015 were reviewed. Data were stratified by assay, geographical location, and patient cohort (general population, patients with HIV, solid-organ transplant recipients, chronic liver disease patients, and individuals in contact with swine/wild animals). Data were pooled using a mixed-effects model. Results: Four hundred thirty-two studies were initially identified, of which 73 studies were included in the analysis. Seroprevalence estimates ranged from 0.6% to 52.5%, increased with age, but were unrelated to gender. General population seroprevalence varied depending on assays: Wantai (WT): 17%, Mikrogen (MG): 10%, MP-diagnostics (MP): 7%, DiaPro: 4%, Abbott 2%. The WT assay reported significantly higher seroprevalence rates across all cohorts (p < 0.001). Individuals in contact with swine/wild animals had significantly higher seroprevalence rates than the general population, irrespective of assay (p < 0.0001). There was no difference between any other cohorts. The highest seroprevalence was observed in France (WT: 32%, MP: 16%) the lowest in Italy (WT: 7.5%, MP 0.9%). Seroprevalence varied between and within countries. The observed heterogeneity was attributed to geographical region (23%), assay employed (23%) and study cohort (7%). Conclusion: Seroprevalcence rates primarily depend on the seroassy that is used, followed by the geographical region and study cohort. Seroprevalence is higher in individuals exposed to swine and/or wild animals, and increases with age. Viruses 2016-08-06 8 8 Review 10.3390/v8080211 211 1999-4915 2016-08-06 doi: 10.3390/v8080211 Johannes Hartl Benjamin Otto Richie Madden Glynn Webb Kathy Woolson Levente Kriston Eik Vettorazzi Ansgar Lohse Harry Dalton Sven Pischke http://www.mdpi.com/1999-4915/8/8/212 Although most hepatitis E virus (HEV) infections are asymptomatic, some can be severe, causing fulminant hepatitis and extra-hepatic manifestations, including neurological and kidney injuries. Chronic HEV infections may also occur in immunocompromised patients. This review describes how our understanding of the pathogenesis of HEV infection has progressed in recent years. Viruses 2016-08-05 8 8 Review 10.3390/v8080212 212 1999-4915 2016-08-05 doi: 10.3390/v8080212 Sébastien Lhomme Olivier Marion Florence Abravanel Sabine Chapuy-Regaud Nassim Kamar Jacques Izopet http://www.mdpi.com/1999-4915/8/8/214 The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo. Viruses 2016-08-04 8 8 Review 10.3390/v8080214 214 1999-4915 2016-08-04 doi: 10.3390/v8080214 Christina Rostad Michael Currier Martin Moore http://www.mdpi.com/1999-4915/8/8/217 The Viruses Editorial Office wishes to notify its readers of corrections in [1].[...] Viruses 2016-08-04 8 8 Erratum 10.3390/v8080217 217 1999-4915 2016-08-04 doi: 10.3390/v8080217 Viruses Office http://www.mdpi.com/1999-4915/8/8/215 Porcine endogenous retroviruses (PERVs) represent a risk factor if porcine cells, tissues, or organs were to be transplanted into human recipients to alleviate the shortage of human transplants; a procedure called xenotransplantation. In contrast to human endogenous retroviruses (HERVs), which are mostly defective and not replication-competent, PERVs are released from normal pig cells and are infectious. PERV-A and PERV-B are polytropic viruses infecting cells of several species, among them humans; whereas PERV-C is an ecotropic virus infecting only pig cells. Virus infection was shown in co-culture experiments, but also in vivo, in the pig, leading to de novo integration of proviruses in certain organs. This was shown by measurement of the copy number per cell, finding different numbers in different organs. In addition, recombinations between PERV-A and PERV-C were observed and the recombinant PERV-A/C were found to be integrated in cells of different organs, but not in the germ line of the animals. Here, the evidence for such in vivo activities of PERVs, including expression as mRNA, protein and virus particles, de novo infection and recombination, will be summarised. These activities make screening of pigs for provirus number and PERV expression level difficult, especially when only blood or ear biopsies are available for analysis. Highly sensitive methods to measure the copy number and the expression level will be required when selecting pigs with low copy number and low expression of PERV as well as when inactivating PERVs using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (CRISPR/Cas) technology. Viruses 2016-08-03 8 8 Review 10.3390/v8080215 215 1999-4915 2016-08-03 doi: 10.3390/v8080215 Joachim Denner http://www.mdpi.com/1999-4915/8/8/213 Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. Viruses 2016-08-03 8 8 Article 10.3390/v8080213 213 1999-4915 2016-08-03 doi: 10.3390/v8080213 Jens Hammerl Claudia Jäckel Erich Lanka Nicole Roschanski Stefan Hertwig http://www.mdpi.com/1999-4915/8/8/206 The 2014 outbreak of Ebola virus (EBOV) in Western Africa highlighted the need for anti-EBOV therapeutics. Clomiphene is a U.S. Food and Drug Administration (FDA)-approved drug that blocks EBOV entry and infection in cells and significantly protects EBOV-challenged mice. As provided, clomiphene is, approximately, a 60:40 mixture of two stereoisomers, enclomiphene and zuclomiphene. The pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which EBOV can persist. Here we compared the ability of clomiphene and its isomers to inhibit EBOV using viral-like particle (VLP) entry and transcription/replication-competent VLP (trVLP) assays. Clomiphene and its isomers inhibited the entry and infection of VLPs and trVLPs with similar potencies. This was demonstrated with VLPs bearing the glycoproteins from three filoviruses (EBOV Mayinga, EBOV Makona, and Marburg virus) and in two cell lines (293T/17 and Vero E6). Visual problems have been noted in EBOV survivors, and viral RNA has been isolated from semen up to nine months post-infection. Since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-EBOV agent, for example, to potentially help ameliorate symptoms in EBOV survivors. Viruses 2016-08-02 8 8 Article 10.3390/v8080206 206 1999-4915 2016-08-02 doi: 10.3390/v8080206 Elizabeth Nelson Alyson Barnes Ronald Wiehle Gregory Fontenot Thomas Hoenen Judith White http://www.mdpi.com/1999-4915/8/8/216 Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. Viruses 2016-08-02 8 8 Review 10.3390/v8080216 216 1999-4915 2016-08-02 doi: 10.3390/v8080216 Maino Tahara Jean-Philippe Bürckert Kazuhiko Kanou Katsumi Maenaka Claude Muller Makoto Takeda http://www.mdpi.com/1999-4915/8/8/209 Influenza remains a serious worldwide public health problem. After infection, viral genomic RNA is replicated in the nucleus and packed into viral ribonucleoprotein, which will then be exported to the cytoplasm via a cellular chromosome region maintenance 1 (CRM1)-dependent pathway for further assembly and budding. However, the nuclear export mechanism of influenza virus remains controversial. Here, we identify cellular nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a novel binding partner of nucleoprotein (NP) that stimulates NP-mediated nuclear export via the CRM1-dependent pathway. NXT1-knockdown cells exhibit decreased viral replication kinetics and nuclear accumulated viral RNA and NP. By contrast, NXT1 overexpression promotes nuclear export of NP in a CRM1-dependent manner. Pull-down assays suggest the formation of an NXT1, NP, and CRM1 complex, and demonstrate that NXT1 binds to the C-terminal region of NP. These findings reveal a distinct mechanism for nuclear export of the influenza virus and identify the NXT1/NP interaction as a potential target for antiviral drug development. Viruses 2016-07-28 8 8 Article 10.3390/v8080209 209 1999-4915 2016-07-28 doi: 10.3390/v8080209 Nopporn Chutiwitoonchai Yoko Aida http://www.mdpi.com/1999-4915/8/8/210 Measles virus is a highly contagious negative strand RNA virus that is transmitted via the respiratory route and causes systemic disease in previously unexposed humans and non-human primates. Measles is characterised by fever and skin rash and usually associated with cough, coryza and conjunctivitis. A hallmark of measles is the transient immune suppression, leading to increased susceptibility to opportunistic infections. At the same time, the disease is paradoxically associated with induction of a robust virus-specific immune response, resulting in lifelong immunity to measles. Identification of CD150 and nectin-4 as cellular receptors for measles virus has led to new perspectives on tropism and pathogenesis. In vivo studies in non-human primates have shown that the virus initially infects CD150+ lymphocytes and dendritic cells, both in circulation and in lymphoid tissues, followed by virus transmission to nectin-4 expressing epithelial cells. The abilities of the virus to cause systemic infection, to transmit to numerous new hosts via droplets or aerosols and to suppress the host immune response for several months or even years after infection make measles a remarkable disease. This review briefly highlights current topics in studies of measles virus host invasion and pathogenesis. Viruses 2016-07-28 8 8 Review 10.3390/v8080210 210 1999-4915 2016-07-28 doi: 10.3390/v8080210 Brigitta Laksono Rory de Vries Stephen McQuaid W. Duprex Rik de Swart http://www.mdpi.com/1999-4915/8/7/208 The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. Viruses 2016-07-23 8 7 Review 10.3390/v8070208 208 1999-4915 2016-07-23 doi: 10.3390/v8070208 Marcio Hedil Richard Kormelink http://www.mdpi.com/1999-4915/8/7/205 The whitefly Bemisia tabaci is a major pest to agricultural crops. It transmits begomoviruses, such as Tomato yellow leaf curl virus (TYLCV), in a circular, persistent fashion. Transcriptome analyses revealed that B. tabaci knottin genes were responsive to various stresses. Upon ingestion of tomato begomoviruses, two of the four knottin genes were upregulated, knot-1 (with the highest expression) and knot-3. In this study, we examined the involvement of B. tabaci knottin genes in relation to TYLCV circulative transmission. Knottins were silenced by feeding whiteflies with knottin dsRNA via detached tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the amounts of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. We suggest that knot-1 protects B. tabaci against deleterious effects caused by TYLCV by limiting the amount of virus associated with the whitefly vector. Viruses 2016-07-22 8 7 Article 10.3390/v8070205 205 1999-4915 2016-07-22 doi: 10.3390/v8070205 Aliza Hariton Shalev Iris Sobol Murad Ghanim Shu-Sheng Liu Henryk Czosnek http://www.mdpi.com/1999-4915/8/7/207 The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV. Viruses 2016-07-22 8 7 Article 10.3390/v8070207 207 1999-4915 2016-07-22 doi: 10.3390/v8070207 Neda Barjesteh Tamiru Alkie Douglas Hodgins Éva Nagy Shayan Sharif http://www.mdpi.com/1999-4915/8/7/204 Effective inactivation of biosafety level 4 (BSL-4) pathogens is vital in order to study these agents safely. Gamma irradiation is a commonly used method for the inactivation of BSL-4 viruses, which among other advantages, facilitates the study of inactivated yet morphologically intact virions. The reported values for susceptibility of viruses to inactivation by gamma irradiation are sometimes inconsistent, likely due to differences in experimental protocols. We analyzed the effects of common sample attributes on the inactivation of a recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein and green fluorescent protein. Using this surrogate virus, we found that sample volume and protein content of the sample modulated viral inactivation by gamma irradiation but that air volume within the sample container and the addition of external disinfectant surrounding the sample did not. These data identify several factors which alter viral susceptibility to inactivation and highlight the usefulness of lower biosafety level surrogate viruses for such studies. Our results underscore the need to validate inactivation protocols of BSL-4 pathogens using "worst-case scenario" procedures to ensure complete sample inactivation. Viruses 2016-07-22 8 7 Article 10.3390/v8070204 204 1999-4915 2016-07-22 doi: 10.3390/v8070204 Adam Hume Joshua Ames Linda Rennick W. Duprex Andrea Marzi John Tonkiss Elke Mühlberger http://www.mdpi.com/1999-4915/8/7/203 In 2007, the United States– Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing. Viruses 2016-07-22 8 7 Article 10.3390/v8070203 203 1999-4915 2016-07-22 doi: 10.3390/v8070203 Jennifer Garver Lauren Weber Eric Vela Mike Anderson Richard Warren Michael Merchlinsky Christopher Houchens James Rogers http://www.mdpi.com/1999-4915/8/7/202 Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle. Viruses 2016-07-21 8 7 Review 10.3390/v8070202 202 1999-4915 2016-07-21 doi: 10.3390/v8070202 Martin Spiegel Teresa Plegge Stefan Pöhlmann http://www.mdpi.com/1999-4915/8/7/201 The ability to accurately and reliably quantify viral infection is essential to basic and translational virology research. Here, we describe a simple and robust automated method for using fluorescence microscopy to estimate the proportion of virally infected cells in a monolayer. We provide details of the automated analysis workflow along with a freely available open-source ImageJ plugin, Infection Counter, for performing image quantification. Using hepatitis C virus (HCV) as an example, we have experimentally verified our method, demonstrating that it is equivalent, if not better, than the established focus-forming assay. Finally, we used Infection Counter to assess the anti-HCV activity of SMBz-CsA, a non-immunosuppressive cyclosporine analogue. Viruses 2016-07-21 8 7 Article 10.3390/v8070201 201 1999-4915 2016-07-21 doi: 10.3390/v8070201 Siân Culley Greg Towers David Selwood Ricardo Henriques Joe Grove http://www.mdpi.com/1999-4915/8/7/200 RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5′ splice site (5′ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5′ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5′ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. Viruses 2016-07-21 8 7 Article 10.3390/v8070200 200 1999-4915 2016-07-21 doi: 10.3390/v8070200 Nancy Mueller Atze Das Ben Berkhout http://www.mdpi.com/1999-4915/8/7/199 Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. Viruses 2016-07-20 8 7 Article 10.3390/v8070199 199 1999-4915 2016-07-20 doi: 10.3390/v8070199 Hongxia Wu Shaopo Zu Xue Sun Yongxiang Liu Jin Tian Liandong Qu http://www.mdpi.com/1999-4915/8/7/197 Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose an important public health problem in their endemic regions. To date, no Food and Drug Administration (FDA)-licensed vaccines are available to combat human arenavirus infections, and current anti-arenaviral drug therapy is limited to an off-label use of ribavirin that is only partially effective. The development of arenavirus reverse genetic approaches has provided investigators with a novel and powerful approach for the study of arenavirus biology including virus–host interactions underlying arenavirus induced disease. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in arenavirus replication and transcription, as well as particle assembly and budding. Likewise, it is now feasible to rescue infectious arenaviruses containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis. The use of reverse genetics approaches has also allowed the generation of recombinant arenaviruses expressing additional genes of interest. These advances in arenavirus molecular genetics have also facilitated the implementation of novel screens to identify anti-arenaviral drugs, and the development of novel strategies for the generation of arenavirus live-attenuated vaccines. In this review, we will summarize the current knowledge on reporter-expressing, replicating-competent arenaviruses harboring reporter genes in different locations of the viral genome and their use for studying and understanding arenavirus biology and the identification of anti-arenaviral drugs to combat these important human pathogens. Viruses 2016-07-20 8 7 Review 10.3390/v8070197 197 1999-4915 2016-07-20 doi: 10.3390/v8070197 Luis Martínez-Sobrido Juan de la Torre http://www.mdpi.com/1999-4915/8/7/198 Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is a malignant human oncovirus belonging to the gamma herpesvirus family. HHV-8 is closely linked to the pathogenesis of Kaposi's sarcoma (KS) and two other B-cell lymphoproliferative diseases: primary effusion lymphoma (PEL) and a plasmablastic variant of multicentric Castleman's disease (MCD). KS is an invasive tumor of endothelial cells most commonly found in untreated HIV-AIDS or immuno-compromised individuals. KS tumors are highly vascularized and have abnormal, excessive neo-angiogenesis, inflammation, and proliferation of infected endothelial cells. KSHV directly induces angiogenesis in an autocrine and paracrine fashion through a complex interplay of various viral and cellular pro-angiogenic and inflammatory factors. KS is believed to originate due to a combination of KSHV's efficient strategies for evading host immune systems and several pro-angiogenic and pro-inflammatory stimuli. In addition, KSHV infection of endothelial cells produces a wide array of viral oncoproteins with transforming capabilities that regulate multiple host-signaling pathways involved in the activation of angiogenesis. It is likely that the cellular-signaling pathways of angiogenesis and lymph-angiogenesis modulate the rate of tumorigenesis induction by KSHV. This review summarizes the current knowledge on regulating KSHV-mediated angiogenesis by integrating the findings reported thus far on the roles of host and viral genes in oncogenesis, recent developments in cell-culture/animal-model systems, and various anti-angiogenic therapies for treating KSHV-related lymphoproliferative disorders. Viruses 2016-07-20 8 7 Review 10.3390/v8070198 198 1999-4915 2016-07-20 doi: 10.3390/v8070198 Pravinkumar Purushothaman Timsy Uppal Roni Sarkar Subhash Verma http://www.mdpi.com/1999-4915/8/7/196 HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA. Viruses 2016-07-18 8 7 Article 10.3390/v8070196 196 1999-4915 2016-07-18 doi: 10.3390/v8070196 Georges Teto Julius Fonsah Claude Tagny Dora Mbanya Emilienne Nchindap Leopoldine Kenmogne Joseph Fokam Dora Njamnshi Charles Kouanfack Alfred Njamnshi Georgette Kanmogne http://www.mdpi.com/1999-4915/8/7/195 Interferons are a group of antiviral cytokines acting as the first line of defense in the antiviral immunity. Here, we describe the antiviral activity of goose type I interferon (IFNα) and type II interferon (IFNγ) against duck plague virus (DPV). Recombinant goose IFNα and IFNγ proteins of approximately 20 kDa and 18 kDa, respectively, were expressed. Following DPV-enhanced green fluorescent protein (EGFP) infection of duck embryo fibroblast cells (DEFs) with IFNα and IFNγ pre-treatment, the number of viral gene copies decreased more than 100-fold, with viral titers dropping approximately 100-fold. Compared to the control, DPV-EGFP cell positivity was decreased by goose IFNα and IFNγ at 36 hpi (3.89%; 0.79%) and 48 hpi (17.05%; 5.58%). In accordance with interferon-stimulated genes being the "workhorse" of IFN activity, the expression of duck myxovirus resistance (Mx) and oligoadenylate synthetases-like (OASL) was significantly upregulated (p < 0.001) by IFN treatment for 24 h. Interestingly, duck cells and goose cells showed a similar trend of increased ISG expression after goose IFNα and IFNγ pretreatment. Another interesting observation is that the positive feedback regulation of type I IFN and type II IFN by goose IFNα and IFNγ was confirmed in waterfowl for the first time. These results suggest that the antiviral activities of goose IFNα and IFNγ can likely be attributed to the potency with which downstream genes are induced by interferon. These findings will contribute to our understanding of the functional significance of the interferon antiviral system in aquatic birds and to the development of interferon-based prophylactic and therapeutic approaches against viral disease. Viruses 2016-07-18 8 7 Article 10.3390/v8070195 195 1999-4915 2016-07-18 doi: 10.3390/v8070195 Hao Zhou Shun Chen Qin Zhou Yunan Wei Mingshu Wang Renyong Jia Dekang Zhu Mafeng Liu Fei Liu Qiao Yang Ying Wu Kunfeng Sun Xiaoyue Chen Anchun Cheng http://www.mdpi.com/1999-4915/8/7/194 The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses. Viruses 2016-07-15 8 7 Review 10.3390/v8070194 194 1999-4915 2016-07-15 doi: 10.3390/v8070194 Katherine Hornak Jean-Marc Lanchy J. Lodmell http://www.mdpi.com/1999-4915/8/7/192 The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV) replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses. Viruses 2016-07-14 8 7 Review 10.3390/v8070192 192 1999-4915 2016-07-14 doi: 10.3390/v8070192 Chris Hellmund Andrew Lever http://www.mdpi.com/1999-4915/8/7/193 Long-terminal repeat (LTR) retrotransposons are transposable genetic elements that replicate intracellularly, and can be considered progenitors of retroviruses. Ty1 and Ty3 are the most extensively characterized LTR retrotransposons whose RNA genomes provide the template for both protein translation and genomic RNA that is packaged into virus-like particles (VLPs) and reverse transcribed. Genomic RNAs are not divided into separate pools of translated and packaged RNAs, therefore their trafficking and packaging into VLPs requires an equilibrium between competing events. In this review, we focus on Ty1 and Ty3 genomic RNA trafficking and packaging as essential steps of retrotransposon propagation. We summarize the existing knowledge on genomic RNA sequences and structures essential to these processes, the role of Gag proteins in repression of genomic RNA translation, delivery to VLP assembly sites, and encapsidation. Viruses 2016-07-14 8 7 Review 10.3390/v8070193 193 1999-4915 2016-07-14 doi: 10.3390/v8070193 Katarzyna Pachulska-Wieczorek Stuart Le Grice Katarzyna Purzycka http://www.mdpi.com/1999-4915/8/7/191 The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. Viruses 2016-07-11 8 7 Article 10.3390/v8070191 191 1999-4915 2016-07-11 doi: 10.3390/v8070191 Marie Kunihiro Hideki Fujii Takuya Miyagi Yoshiaki Takahashi Reiko Tanaka Takuya Fukushima Aftab Ansari Yuetsu Tanaka http://www.mdpi.com/1999-4915/8/7/190 The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. Viruses 2016-07-08 8 7 Article 10.3390/v8070190 190 1999-4915 2016-07-08 doi: 10.3390/v8070190 Katharina Hipp Benjamin Schäfer Gabi Kepp Holger Jeske http://www.mdpi.com/1999-4915/8/7/188 Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated badnavirus sequences in some yam genomes. To date, only two badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4–7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed badnavirus infections occur relatively often in West African yam germplasm. Viruses 2016-07-07 8 7 Article 10.3390/v8070188 188 1999-4915 2016-07-07 doi: 10.3390/v8070188 Moritz Bömer Aliyu Turaki Gonçalo Silva P. Kumar Susan Seal http://www.mdpi.com/1999-4915/8/7/189 Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. Viruses 2016-07-07 8 7 Article 10.3390/v8070189 189 1999-4915 2016-07-07 doi: 10.3390/v8070189 Marcela Sabino Cunha Thatiane Lima Sampaio B. Peterlin Luciana Jesus da Costa http://www.mdpi.com/1999-4915/8/7/185 The West Africa Ebola virus (EBOV) outbreak has highlighted the need for effective disinfectants capable of reducing viral load in a range of sample types, equipment and settings. Although chlorine-based products are widely used, they can also be damaging to equipment or apparatus that needs continuous use such as aircraft use for transportation of infected people. Two aircraft cleaning solutions were assessed alongside two common laboratory disinfectants in a contact kill assay with EBOV on two aircraft relevant materials representative of a porous and non-porous surface. A decimal log reduction of viral titre of 4 is required for a disinfectant to be deemed effective and two of the disinfectants fulfilled this criteria under the conditions tested. One product, Ardrox 6092, was found to perform similarly to sodium hypochlorite, but as it does not have the corrosive properties of sodium hypochlorite, it could be an alternative disinfectant solution to be used for decontamination of EBOV on sensitive apparatus. Viruses 2016-07-07 8 7 Article 10.3390/v8070185 185 1999-4915 2016-07-07 doi: 10.3390/v8070185 Sophie Smither Amanda Phelps Lin Eastaugh Sarah Ngugi Lyn O'Brien Andrew Dutch Mark Lever http://www.mdpi.com/1999-4915/8/7/187 Ranaviruses (Iridoviridae) are large DNA viruses that are causing emerging infectious diseases at an alarming rate in both wild and captive cold blood vertebrate species all over the world. Although the general biology of these viruses that presents some similarities with poxvirus is characterized, many aspects of their replication cycles, host cell interactions and evolution still remain largely unclear, especially in vivo. Over several years, strategies to generate site-specific ranavirus recombinant, either expressing fluorescent reporter genes or deficient for particular viral genes, have been developed. We review here these strategies, the main ranavirus recombinants characterized and their usefulness for in vitro and in vivo studies. Viruses 2016-07-06 8 7 Review 10.3390/v8070187 187 1999-4915 2016-07-06 doi: 10.3390/v8070187 Jacques Robert James Jancovich http://www.mdpi.com/1999-4915/8/7/186 Knowing about the genes involved in immunity, and being able to identify the factors influencing their expressions, helps in gaining awareness of the immune processes. The qPCR method is a useful gene expression analysis tool, but studies on immune system genes are still limited, especially on the caprine immune system. Caprine arthritis encephalitis, a disease caused by small ruminant lentivirus (SRLV), causes economic losses in goat breeding, and there is no therapy against SRLV. The results of studies on vaccines against other viruses are promising. Moreover, the Marker-Assisted Selection strategy against SRLV is possible, as has been shown in sheep breeding. However, there are still many gaps in our knowledge on the caprine immune response to infection. All types of cytokines play pivotal roles in immunity, and SRLV infection influences the expression of many cytokines in different types of cells. This information encouraged the authors to examine the results of studies conducted on SRLV and other viral infections, with an emphasis on the expression of cytokine genes. This review attempts to summarize the results of studies on the expression of cytokines in the context of the SRLV infection. Viruses 2016-07-05 8 7 Review 10.3390/v8070186 186 1999-4915 2016-07-05 doi: 10.3390/v8070186 Justyna Jarczak Jarosław Kaba Daria Reczyńska Emilia Bagnicka http://www.mdpi.com/1999-4915/8/7/183 Viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. Avirulent NDV strains LaSota and B1 have long track records of safety and efficacy. Therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. NDV replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. As a vaccine vector, NDV can accommodate foreign sequences with a good degree of stability and as a RNA virus, there is limited possibility for recombination with host cell DNA. Using NDV as a vaccine vector in humans offers several advantages over other viral vaccine vectors. NDV is safe in humans due to host range restriction and there is no pre-existing antibody to NDV in the human population. NDV is antigenically distinct from common human pathogens. NDV replicates to high titer in a cell line acceptable for human vaccine development. Therefore, NDV is an attractive vaccine vector for human pathogens for which vaccines are currently not available. NDV is also an attractive vaccine vector for animal pathogens. Viruses 2016-07-04 8 7 Review 10.3390/v8070183 183 1999-4915 2016-07-04 doi: 10.3390/v8070183 Shin-Hee Kim Siba Samal http://www.mdpi.com/1999-4915/8/7/184 Similar to other viruses, coronavirus infection triggers cellular stress responses in infected host cells. The close association of coronavirus replication with the endoplasmic reticulum (ER) results in the ER stress responses, which impose a challenge to the viruses. Viruses, in turn, have come up with various mechanisms to block or subvert these responses. One of the ER stress responses is inhibition of the global protein synthesis to reduce the amount of unfolded proteins inside the ER lumen. Viruses have evolved the capacity to overcome the protein translation shutoff to ensure viral protein production. Here, we review the strategies exploited by coronavirus to modulate cellular stress response pathways. The involvement of coronavirus-induced stress responses and translational control in viral pathogenesis will also be briefly discussed. Viruses 2016-07-04 8 7 Review 10.3390/v8070184 184 1999-4915 2016-07-04 doi: 10.3390/v8070184 To Fung Ying Liao Ding Liu http://www.mdpi.com/1999-4915/8/7/182 Among the components of the RNA silencing pathway in plants, RNA-dependent RNA polymerases (RDRs) play fundamental roles in antiviral defence. Here, we demonstrate that the Nicotiana benthamiana RDR6 is involved in defence against the bipartite crinivirus (genus Crinivirus, family Closteroviridae) Tomato chlorosis virus (ToCV). Additionally, by producing a p22-deficient ToCV infectious mutant clone (ToCVΔp22), we studied the role of this viral suppressor of RNA silencing in viral infection in both wild-type and RDR6-silenced N. benthamiana (NbRDR6i) plants. We demonstrate that p22 is dispensable for the replication of ToCV, where RDR6 appears not to have any effect. Furthermore, the finding that ToCV∆p22 systemic accumulation was impaired in wild-type N. benthamiana but not in NbRDR6i plants suggests a role for p22 in counteracting an RDR6-mediated antiviral response of the plant during systemic infection. Viruses 2016-06-28 8 7 Brief Report 10.3390/v8070182 182 1999-4915 2016-06-28 doi: 10.3390/v8070182 Yazmín Landeo-Ríos Jesús Navas-Castillo Enrique Moriones M. Cañizares http://www.mdpi.com/1999-4915/8/7/180 After viral infection, host cells respond by mounting an anti-viral stress response in order to create a hostile atmosphere for viral replication, leading to the shut-off of mRNA translation (protein synthesis) and the assembly of RNA granules. Two of these RNA granules have been well characterized in yeast and mammalian cells, stress granules (SGs), which are translationally silent sites of RNA triage and processing bodies (PBs), which are involved in mRNA degradation. This review discusses the role of these RNA granules in the evasion of anti-viral stress responses through virus-induced remodeling of cellular ribonucleoproteins (RNPs). Viruses 2016-06-28 8 7 Review 10.3390/v8070180 180 1999-4915 2016-06-28 doi: 10.3390/v8070180 Natalia Poblete-Durán Yara Prades-Pérez Jorge Vera-Otarola Ricardo Soto-Rifo Fernando Valiente-Echeverría http://www.mdpi.com/1999-4915/8/7/181 Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV. Viruses 2016-06-28 8 7 Article 10.3390/v8070181 181 1999-4915 2016-06-28 doi: 10.3390/v8070181 Cheng-Lin Deng Si-Qing Liu Dong-Gen Zhou Lin-Lin Xu Xiao-Dan Li Pan-Tao Zhang Peng-Hui Li Han-Qing Ye Hong-Ping Wei Zhi-Ming Yuan Cheng-Feng Qin Bo Zhang http://www.mdpi.com/1999-4915/8/7/179 Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. Viruses 2016-06-23 8 7 Review 10.3390/v8070179 179 1999-4915 2016-06-23 doi: 10.3390/v8070179 Michael Breen Aitor Nogales Steven Baker Luis Martínez-Sobrido http://www.mdpi.com/1999-4915/8/6/177 Badnaviruses (Family: Caulimoviridae; Genus: Badnavirus) are non-enveloped bacilliform DNA viruses with a monopartite genome containing about 7.2 to 9.2 kb of dsDNA with three to seven open reading frames. They are transmitted by mealybugs and a few species by aphids in a semi-persistent manner. They are one of the most important plant virus groups and have emerged as serious pathogens affecting the cultivation of several horticultural crops in the tropics, especially banana, black pepper, cocoa, citrus, sugarcane, taro, and yam. Some badnaviruses are also known as endogenous viruses integrated into their host genomes and a few such endogenous viruses can be awakened, e.g., through abiotic stress, giving rise to infective episomal forms. The presence of endogenous badnaviruses poses a new challenge for the fool-proof diagnosis, taxonomy, and management of the diseases. The present review aims to highlight emerging disease problems, virus characteristics, transmission, and diagnosis of badnaviruses. Viruses 2016-06-22 8 6 Review 10.3390/v8060177 177 1999-4915 2016-06-22 doi: 10.3390/v8060177 Alangar Bhat Thomas Hohn Ramasamy Selvarajan http://www.mdpi.com/1999-4915/8/6/172 The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (RifR) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the RifR mutants remained PS and localized to the Rif binding pocket in RNAP, but a subset acquired a PR phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One PR mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP. Viruses 2016-06-22 8 6 Article 10.3390/v8060172 172 1999-4915 2016-06-22 doi: 10.3390/v8060172 Sidney Hayes Wen Wang Karthic Rajamanickam Audrey Chu Anirban Banerjee Connie Hayes http://www.mdpi.com/1999-4915/8/6/175 Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer. Viruses 2016-06-22 8 6 Article 10.3390/v8060175 175 1999-4915 2016-06-22 doi: 10.3390/v8060175 Molly Bristol Xu Wang Nathan Smith Minkyeong Son Michael Evans Iain Morgan http://www.mdpi.com/1999-4915/8/6/178 The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems. Viruses 2016-06-22 8 6 Review 10.3390/v8060178 178 1999-4915 2016-06-22 doi: 10.3390/v8060178 Kristina Schmidt Elke Mühlberger http://www.mdpi.com/1999-4915/8/6/174 The genus Phlebovirus of the family Bunyaviridae contains a number of emerging virus species which pose a threat to both human and animal health. Most prominent members include Rift Valley fever virus (RVFV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV), Toscana virus (TOSV), Punta Toro virus (PTV), and the two new members severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV). The nonstructural protein NSs is well established as the main phleboviral virulence factor in the mammalian host. NSs acts as antagonist of the antiviral type I interferon (IFN) system. Recent progress in the elucidation of the molecular functions of a growing list of NSs proteins highlights the astonishing variety of strategies employed by phleboviruses to evade the IFN system. Viruses 2016-06-22 8 6 Review 10.3390/v8060174 174 1999-4915 2016-06-22 doi: 10.3390/v8060174 Jennifer Wuerth Friedemann Weber http://www.mdpi.com/1999-4915/8/6/176 While addition of the first-approved protease inhibitors (PIs), telaprevir and boceprevir, to pegylated interferon (PEG-IFN) and ribavirin (RBV) combination therapy significantly increased sustained virologic response (SVR) rates, PI-based triple therapy for the treatment of chronic hepatitis C virus (HCV) infection was prone to the emergence of resistant viral variants. Meanwhile, multiple direct acting antiviral agents (DAAs) targeting either the HCV NS3/4A protease, NS5A or NS5B polymerase have been approved and these have varying potencies and distinct propensities to provoke resistance. The pre-clinical in vivo assessment of drug efficacy and resistant variant emergence underwent a great evolution over the last decade. This field had long been hampered by the lack of suitable small animal models that robustly support the entire HCV life cycle. In particular, chimeric mice with humanized livers (humanized mice) and chimpanzees have been instrumental for studying HCV inhibitors and the evolution of drug resistance. In this review, we present the different in vivo HCV infection models and discuss their applicability to assess HCV therapy response and emergence of resistant variants. Viruses 2016-06-22 8 6 Review 10.3390/v8060176 176 1999-4915 2016-06-22 doi: 10.3390/v8060176 Ahmed Mesalam Koen Vercauteren Philip Meuleman http://www.mdpi.com/1999-4915/8/6/173 The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells. Viruses 2016-06-21 8 6 Article 10.3390/v8060173 173 1999-4915 2016-06-21 doi: 10.3390/v8060173 Amr Gamil Cheng Xu Stephen Mutoloki Øystein Evensen http://www.mdpi.com/1999-4915/8/6/165 Influenza A viruses (IAVs) harbor a segmented RNA genome that is organized into eight distinct viral ribonucleoprotein (vRNP) complexes. Although a segmented genome may be a major advantage to adapt to new host environments, it comes at the cost of a highly sophisticated genome packaging mechanism. Newly synthesized vRNPs conquer the cellular endosomal recycling machinery to access the viral budding site at the plasma membrane. Genome packaging sequences unique to each RNA genome segment are thought to be key determinants ensuring the assembly and incorporation of eight distinct vRNPs into progeny viral particles. Recent studies using advanced fluorescence microscopy techniques suggest the formation of vRNP sub-bundles (comprising less than eight vRNPs) during their transport on recycling endosomes. The formation of such sub-bundles might be required for efficient packaging of a bundle of eight different genomes segments at the budding site, further highlighting the complexity of IAV genome packaging. Viruses 2016-06-17 8 6 Review 10.3390/v8060165 165 1999-4915 2016-06-17 doi: 10.3390/v8060165 Sebastian Giese Hardin Bolte Martin Schwemmle http://www.mdpi.com/1999-4915/8/6/171 Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases, such as adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/Tropic spastic paraparesis (HAM/TSP). As a retrovirus, its life cycle includes a step where HTLV-1 is integrated into the host genomic DNA and forms proviral DNA. In the chronic phase of the infection, HTLV‑1 is known to proliferate as a provirus via the mitotic division of the infected host cells. There are generally tens of thousands of infected clones within an infected individual. They exist not only in peripheral blood, but also in various lymphoid organs. Viral proteins encoded in HTLV-1 genome play a role in the proliferation and survival of the infected cells. As is the case with other chronic viral infections, HTLV-1 gene expression induces the activation of the host immunity against the virus. Thus, the transcription from HTLV-1 provirus needs to be controlled in order to evade the host immune surveillance. There should be a dynamic and complex regulation in vivo, where an equilibrium between viral antigen expression and host immune surveillance is achieved. The mechanisms regulating viral gene expression from the provirus are a key to understanding the persistent/latent infection with HTLV-1 and its pathogenesis. In this article, we would like to review our current understanding on this topic. Viruses 2016-06-16 8 6 Review 10.3390/v8060171 171 1999-4915 2016-06-16 doi: 10.3390/v8060171 Paola Miyazato Misaki Matsuo Hiroo Katsuya Yorifumi Satou