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Table of Contents for this page:

  • Current Issue
  • Current Issue of Viruses


  • Viruses, Vol. 7, Pages 5243-5256: HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

  • Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

  • Viruses, Vol. 7, Pages 5225-5242: Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97

  • A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.

  • Viruses, Vol. 7, Pages 5206-5224: Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance

  • The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development, and direct-acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.

  • Viruses, Vol. 7, Pages 5191-5205: Differentially-Expressed Pseudogenes in HIV-1 Infection

  • Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these“functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

  • Viruses, Vol. 7, Pages 5172-5190: Filoviruses: One of These Things is (not) Like the Other

  • The family Filoviridae contains several of the most deadly pathogens known to date and the current Ebola virus disease (EVD) outbreak in Western Africa, due to Ebola virus (EBOV) infection, highlights the need for active and broad research into filovirus pathogenesis. However, in comparison, the seven other known filovirus family members are significantly understudied. Many of these, including Marburgviruses and Ebolaviruses other than EBOV, are also highly virulent and fully capable of causing widespread epidemics. This review places the focus on these non-EBOV filoviruses, including known immunological and pathological data. The available animal models, research tools and currently available therapeutics will also be discussed along with an emphasis in the large number of current gaps in knowledge of these less highlighted filoviruses. It is evident that much research is yet to be done in order to bring the non-EBOV filovirus field to the forefront of current research and, importantly, to the development of more effective vaccines and therapeutics to combat potential future outbreaks.

  • Viruses, Vol. 7, Pages 5169-5171: Viroporins: Structures and functions beyond cell membrane permeabilization

  • Viroporins represent an interesting group of viral proteins that exhibit two sets of functions. First, they participate in several viral processes that are necessary for efficient production of virus progeny. [...]

  • Viruses, Vol. 7, Pages 5155-5168: Interferon-Free Hepatitis C Treatment before and after Liver Transplantation: The Role of HCV Drug Resistance

  • Hepatitis C virus (HCV) infection is one of the leading causes of end-stage liver disease and the main indication for liver transplantation (LT) in most countries. All patients who undergo LT with detectable serum HCV RNA experience graft reinfection progressing to cirrhosis within five years in 20% to 30% of them. Obtaining a sustained virological response (SVR) greatly improves overall and graft survival. Until 2011, standard antiviral therapy using PEGylated interferon (PEG-IFN) and ribavirin (RBV) was the only effective therapy, with an SVR rate around 30% in this setting. For patients infected with genotype 1, first generation NS3/4A protease inhibitors (PIs), boceprevir (BOC) or telaprevir (TVR), associated with PEG-IFN and RBV for 48 weeks have increased the SVR rates to 60% in non-transplant patients. However, tolerability and drug-drug interactions with calcineurin inhibitors (CNI) are both limiting factors of their use in the liver transplant setting. Over recent years, the efficacy of antiviral C therapy has improved dramatically using new direct-acting antiviral (DAA) agents without PEG-IFN and/or RBV, leading to SVR rates over 90% in non-transplant patients. Results available for transplant patients showed a better efficacy and tolerability and less drug-drug interactions than with first wave PIs. However, some infrequent cases of viral resistance have been reported using PIs or NS5A inhibitors pre- or post-LT that can lead to difficulties in the management of these patients.

  • Viruses, Vol. 7, Pages 5145-5154: Tight Junctions Go Viral!

  • Tight junctions (TJs) are highly specialized membrane domains involved in many important cellular processes such as the regulation of the passage of ions and macromolecules across the paracellular space and the establishment of cell polarity in epithelial cells. Over the past few years there has been increasing evidence that different components of the TJs can be hijacked by viruses in order to complete their infectious cycle. Viruses from at least nine different families of DNA and RNA viruses have been reported to use TJ proteins in their benefit. For example, TJ proteins such as JAM-A or some members of the claudin family of proteins are used by members of the Reoviridae family and hepatitis C virus as receptors or co-receptors during their entry into their host cells. Reovirus, in addition, takes advantage of the TJ protein Junction Adhesion Molecule-A (JAM-A) to achieve its hematogenous dissemination. Some other viruses are capable of regulating the expression or the localization of TJ proteins to induce cell transformation or to improve the efficiency of their exit process. This review encompasses the importance of TJs for viral entry, replication, dissemination, and egress, and makes a clear statement of the importance of studying these proteins to gain a better understanding of the replication strategies used by viruses that infect epithelial and/or endothelial cells.

  • Viruses, Vol. 7, Pages 5133-5144: Preventive Activity against Influenza (H1N1) Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice

  • The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.

  • Viruses, Vol. 7, Pages 5115-5132: Conformational Masking and Receptor-Dependent Unmasking of Highly Conserved Env Epitopes Recognized by Non-Neutralizing Antibodies That Mediate Potent ADCC against HIV-1

  • The mechanism of antibody-mediated protection is a major focus of HIV-1 vaccine development and a significant issue in the control of viremia. Virus neutralization, Fc-mediated effector function, or both, are major mechanisms of antibody-mediated protection against HIV-1, although other mechanisms, such as virus aggregation, are known. The interplay between virus neutralization and Fc-mediated effector function in protection against HIV-1 is complex and only partially understood. Passive immunization studies using potent broadly neutralizing antibodies (bnAbs) show that both neutralization and Fc-mediated effector function provides the widest dynamic range of protection; however, a vaccine to elicit these responses remains elusive. By contrast, active immunization studies in both humans and non-human primates using HIV-1 vaccine candidates suggest that weakly neutralizing or non-neutralizing antibodies can protect by Fc-mediated effector function, albeit with a much lower dynamic range seen for passive immunization with bnAbs. HIV-1 has evolved mechanisms to evade each type of antibody-mediated protection that must be countered by a successful AIDS vaccine. Overcoming the hurdles required to elicit bnAbs has become a major focus of HIV-1 vaccine development. Here, we discuss a less studied problem, the structural basis of protection (and its evasion) by antibodies that protect only by potent Fc-mediated effector function.

  • Viruses, Vol. 7, Pages 5084-5114: Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

  • Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called“tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei.

  • Viruses, Vol. 7, Pages 5066-5083: Exosome Biogenesis, Regulation, and Function in Viral Infection

  • Exosomes are extracellular vesicles released upon fusion of multivesicular bodies(MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) duringthe process of MVB formation. Exosomes were shown to contain selectively sorted functionalproteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in thephysiology of the healthy and diseased organism. Challenges in the field include the identificationof mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles andthe understanding of the underlying processes directing MVBs for degradation or fusion with theplasma membrane. The investigation into the formation and roles of exosomes in viral infection is inits early years. Although still controversial, exosomes can, in principle, incorporate any functionalfactor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation.This review initially focuses on the composition and biogenesis of exosomes. It then explores theregulatory mechanisms underlying their biogenesis. Exosomes are part of the endocytic system,which is tightly regulated and able to respond to several stimuli that lead to alterations in thecomposition of its sub-compartments. We discuss the current knowledge of how these changesaffect exosomal release. We then summarize how different viruses exploit specific proteins ofendocytic sub-compartments and speculate that it could interfere with exosome function, althoughno direct link between viral usage of the endocytic system and exosome release has yet beenreported. Many recent reports have ascribed functions to exosomes released from cells infectedwith a variety of animal viruses, including viral spread, host immunity, and manipulation of themicroenvironment, which are discussed. Given the ever-growing roles and importance of exosomesin viral infections, understanding what regulates their composition and levels, and defining theirfunctions will ultimately provide additional insights into the virulence and persistence of infections.

  • Viruses, Vol. 7, Pages 5040-5065: Recent Progress in Therapeutic Treatments and Screening Strategies for the Prevention and Treatment of HPV-Associated Head and Neck Cancer

  • The rise in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has elicited significant interest in the role of high-risk HPV in tumorigenesis. Because patients with HPV-positive HNSCC have better prognoses than do their HPV-negative counterparts, current therapeutic strategies for HPV+ HNSCC are increasingly considered to be overly aggressive, highlighting a need for customized treatment guidelines for this cohort. Additional issues include the unmet need for a reliable screening strategy for HNSCC, as well as the ongoing assessment of the efficacy of prophylactic vaccines for the prevention of HPV infections in the head and neck regions. This review also outlines a number of emerging prospects for therapeutic vaccines, as well as for targeted, molecular-based therapies for HPV-associated head and neck cancers. Overall, the future for developing novel and effective therapeutic agents for HPV-associated head and neck tumors is promising; continued progress is critical in order to meet the challenges posed by the growing epidemic.

  • Viruses, Vol. 7, Pages 5018-5039: Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1–6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance

  • Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46%) and predominantly found to be under negative selective pressure, suggestingmainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be genotype-specific or geographically tailored.

  • Viruses, Vol. 7, Pages 5016-5017: Jan van der Noordaa (1934–2015); A Virologist Pur Sang

  • Our loyal friend and colleague, Jan van der Noordaa, passed away unexpectedly at the age of 80 on the evening of 17 June 2015. [...]

  • Viruses, Vol. 7, Pages 4997-5015: High Rate of Simian Immunodeficiency Virus (SIV) Infections in Wild Chimpanzees in Northeastern Gabon

  • The emergence of HIV-1 groups M, N, O, and P is the result of four independent cross-species transmissions between chimpanzees (cpz) and gorillas (gor) from central/south Cameroon and humans respectively. Although the first two SIVcpz were identified in wild-born captive chimpanzees in Gabon in 1989, no study has been conducted so far in wild chimpanzees in Gabon. To document the SIVcpz infection rate, genetic diversity, and routes of virus transmission, we analyzed 1458 faecal samples collected in 16 different locations across the country, and we conducted follow-up missions in two of them. We found 380 SIV antibody positive samples in 6 different locations in the north and northeast. We determined the number of individuals collected by microsatellite analysis and obtained an adjusted SIV prevalence of 39.45%. We performed parental analysis to investigate viral spread between and within communities and found that SIVs were epidemiologically linked and were transmitted by both horizontal and vertical routes. We amplified pol and gp41 fragments and obtained 57 new SIVcpzPtt strains from three sites. All strains, but one, clustered together within a specific phylogeographic clade. Given that these SIV positive samples have been collected nearby villages and that humans continue to encroach in ape’s territories, the emergence of a new HIV in this area needs to be considered.

  • Viruses, Vol. 7, Pages 4978-4996: Alterations of Nuclear Architecture and Epigenetic Signatures during African Swine Fever Virus Infection

  • Viral interactions with host nucleus have been thoroughly studied, clarifying molecular mechanisms and providing new antiviral targets. Considering that African swine fever virus (ASFV) intranuclear phase of infection is poorly understood, viral interplay with subnuclear domains and chromatin architecture were addressed. Nuclear speckles, Cajal bodies, and promyelocytic leukaemia nuclear bodies (PML-NBs) were evaluated by immunofluorescence microscopy and Western blot. Further, efficient PML protein knockdown by shRNA lentiviral transduction was used to determine PML-NBs relevance during infection. Nuclear distribution of different histone H3 methylation marks at lysine’s 9, 27 and 36, heterochromatin protein 1 isoforms (HP1α, HPβ and HPγ) and several histone deacetylases (HDACs) were also evaluated to assess chromatin status of the host. Our results reveal morphological disruption of all studied subnuclear domains and severe reduction of viral progeny in PML-knockdown cells. ASFV promotes H3K9me3 and HP1β foci formation from early infection, followed by HP1α and HDAC2 nuclear enrichment, suggesting heterochromatinization of host genome. Finally, closeness between DNA damage response factors, disrupted PML-NBs, and virus-induced heterochromatic regions were identified. In sum, our results demonstrate that ASFV orchestrates spatio-temporal nuclear rearrangements, changing subnuclear domains, relocating Ataxia Telangiectasia Mutated Rad-3 related (ATR)-related factors and promoting heterochromatinization, probably controlling transcription, repressing host gene expression, and favouring viral replication.

  • Viruses, Vol. 7, Pages 4960-4977: Anti-HBV Drugs: Progress, Unmet Needs, and New Hope

  • Approximately 240 million people worldwide are chronically infected with hepatitis B virus (HBV), which represents a significant challenge to public health. The current goal in treating chronic HBV infection is to block progression of HBV-related liver injury and inflammation to end-stage liver diseases, including cirrhosis and hepatocellular carcinoma, because we are unable to eliminate chronic HBV infection. Available therapies for chronic HBV infection mainly include nucleos/tide analogues (NAs), non-NAs, and immunomodulatory agents. However, none of them is able to clear chronic HBV infection. Thus, a new generation of anti-HBV drugs is urgently needed. Progress has been made in the development and testing of new therapeutics against chronic HBV infection. This review aims to summarize the state of the art in new HBV drug research and development and to forecast research and development trends and directions in the near future.

  • Viruses, Vol. 7, Pages 4945-4959: Determinants of Disease Phenotype Differences Caused by Closely-Related Isolates of Begomovirus Betasatellites Inoculated with the Same Species of Helper Virus

  • Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus associated with different betasatellites. In this study, we investigate two different isolates of Tomato yellow leaf curl China betasatellite (TYLCCNB) to determine what features of the viral genome are required for induction of characteristic phenotypic differences between closely-related betasatellite. When co-agroinoculated with TYLCCNV into Nicotiana spp. and tomato plants, TYLCCNB-Y25 induced only leaf curling on all hosts, while TYLCCNB-Y10 also induced enations, vein yellowing, and shoot distortions. Further assays showed thatβC1 of TYLCCNB-Y25 differs from that of TYLCCNB-Y10 in symptom induction and transcriptional modulating. Hybrid satellites were constructed in which the βC1 gene or 200 nt partial promoter-like fragment upstream of the βC1 were exchanged. Infectivity assays showed that a TYLCCNB-Y25 hybrid with the intact TYLCCNB-Y10 βC1 gene was able to induce vein yellowing, shoot distortions, and a reduced size and number of enations. A TYLCCNB-Y10 hybrid with the intact TYLCCNB-Y25 βC1 gene produced only leaf curling. In contrast, the TYLCCNB-Y25 and TYLCCNB-Y10 hybrids with swapped partial promoter-like regions had little effect on the phenotypes induced by wild-type betasatellites. Further experiments showed that the TYLCCNB-Y25 hybrid carrying the C-terminal region of TYLCCNB-Y10 βC1 induced TYLCCNB-Y10-like symptoms. These findings indicate that the βC1 protein is the major symptom determinant and that the C-terminal region of βC1 plays an important role in symptom induction.

  • Viruses, Vol. 7, Pages 4929-4944: Clinical Implications of Antiviral Resistance in Influenza

  • Influenza is a major cause of severe respiratory infections leading to excessive hospitalizations and deaths globally; annual epidemics, pandemics, and sporadic/endemic avian virus infections occur as a result of rapid, continuous evolution of influenza viruses. Emergence of antiviral resistance is of great clinical and public health concern. Currently available antiviral treatments include four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir, laninamivir), M2-inibitors (amantadine, rimantadine), and a polymerase inhibitor (favipiravir). In this review, we focus on resistance issues related to the use of neuraminidase inhibitors (NAIs). Data on primary resistance, as well as secondary resistance related to NAI exposure will be presented. Their clinical implications, detection, and novel therapeutic options undergoing clinical trials are discussed.

  • Viruses, Vol. 7, Pages 4911-4928: Insect-Specific Virus Discovery: Significance for the Arbovirus Community

  • Arthropod-borne viruses (arboviruses), especially those transmitted by mosquitoes, are a significant cause of morbidity and mortality in humans and animals worldwide. Recent discoveries indicate that mosquitoes are naturally infected with a wide range of other viruses, many within taxa occupied by arboviruses that are considered insect-specific. Over the past ten years there has been a dramatic increase in the literature describing novel insect-specific virus detection in mosquitoes, which has provided new insights about viral diversity and evolution, including that of arboviruses. It has also raised questions about what effects the mosquito virome has on arbovirus transmission. Additionally, the discovery of these new viruses has generated interest in their potential use as biological control agents as well as novel vaccine platforms. The arbovirus community will benefit from the growing database of knowledge concerning these newly described viral endosymbionts, as their impacts will likely be far reaching.

  • Viruses, Vol. 7, Pages 4894-4910: Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection

  • Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced aamp;amp;gt;90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

  • Viruses, Vol. 7, Pages 4873-4893: Structural Conservation and Functional Diversity of the Poxvirus Immune Evasion (PIE) Domain Superfamily

  • Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Structural analysis of four of these seemingly unrelated proteins revealed the recurrent use of a conserved beta-sandwich fold that has not been observed in any eukaryotic or prokaryotic protein. Herein we propose to call this unique structural scaffolding the PIE (Poxvirus Immune Evasion) domain. PIE domain containing proteins are abundant in chordopoxvirinae, with our analysis identifying 20 likely PIE subfamilies among 33 representative genomes spanning 7 genera. For example, cowpox strain Brighton Red appears to encode 10 different PIEs: vCCI, A41, C8, M2, T4 (CPVX203), and the SECRET proteins CrmB, CrmD, SCP-1, SCP-2, and SCP-3. Characterized PIE proteins all appear to be nonessential for virus replication, and all contain signal peptides for targeting to the secretory pathway. The PIE subfamilies differ primarily in the number, size, and location of structural embellishments to the beta-sandwich core that confer unique functional specificities. Reported ligands include chemokines, GM-CSF, IL-2, MHC class I, and glycosaminoglycans. We expect that the list of ligands and receptors engaged by the PIE domain will grow as we come to better understand how this versatile structural architecture can be tailored to manipulate host responses to infection.

  • Viruses, Vol. 7, Pages 4854-4872: Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

  • Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways.

  • Viruses, Vol. 7, Pages 4836-4853: Bio-Control of Salmonella Enteritidis in Foods Using Bacteriophages

  • Two lytic phages, vB_SenM-PA13076 (PA13076) and vB_SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages in liquid culture showed 104 multiplicity of infection (MOI) was the best in inhibiting bacteria, with PC2184 exhibiting more activity than PA13076. The two phages exhibited broad host range within the genus Salmonella. Phage PA13076 and PC2184 had a lytic effect on 222 (71.4%) and 298 (95.8%) of the 311 epidemic Salmonella isolates, respectively. We tested the effectiveness of phage PA13076 and PC2184 as well as a cocktail combination of both in three different foods (chicken breast, pasteurized whole milk and Chinese cabbage) contaminated with SE. Samples were spiked with 1× 104 CFU individual SE or a mixture of strains (ATCC13076 and CVCC2184), then treated with 1 × 108 PFU individual phage or a two phage cocktail, and incubated at 4 °C or 25 °C for 5 h. In general, the inhibitory effect of phage and phage cocktail was better at 4 °C than that at 25 °C, whereas the opposite result was observed in Chinese cabbage, and phage cocktail was better than either single phage. A significant reduction in bacterial numbers (1.5–4 log CFU/sample, p aamp;amp;lt; 0.05) was observed in all tested foods. The two phages on the three food samples were relatively stable,especially at 4 ºC, with the phages exhibiting the greatest stability in milk. Our research shows that our phages have potential effectiveness as a bio-control agent of Salmonella in foods.

  • Viruses, Vol. 7, Pages 4826-4835: New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging

  • Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

  • Viruses, Vol. 7, Pages 4800-4825: Cloak and Dagger: Alternative Immune Evasion and Modulation Strategies of Poxviruses

  • As all viruses rely on cellular factors throughout their replication cycle, to be successful they must evolve strategies to evade and/or manipulate the defence mechanisms employed by the host cell. In addition to their expression of a wide array of host modulatory factors, several recent studies have suggested that poxviruses may have evolved unique mechanisms to shunt or evade host detection. These potential mechanisms include mimicry of apoptotic bodies by mature virions (MVs), the use of viral sub-structures termed lateral bodies for the packaging and delivery of host modulators, and the formation of a second,“cloaked” form of infectious extracellular virus (EVs). Here we discuss these various strategies and how they may facilitate poxvirus immune evasion. Finally we propose a model for the exploitation of the cellular exosome pathway for the formation of EVs.

  • Viruses, Vol. 7, Pages 4783-4799: Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

  • A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA) were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79); the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus) presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

  • Viruses, Vol. 7, Pages 4772-4782: Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

  • Transcription activator–like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses.

  • Viruses, Vol. 7, Pages 4756-4771: The Antiviral Effect of Baicalin on Enterovirus 71 In Vitro

  • Baicalin is a flavonoid compound extracted from Scutellaria roots that has been reported to possess antibacterial, anti-inflammatory, and antiviral activities. However, the antiviral effect of baicalin on enterovirus 71 (EV71) is still unknown. In this study, we found that baicalin showed inhibitory activity on EV71 infection and was independent of direct virucidal or prophylactic effect and inhibitory viral absorption. The expressions of EV71/3D mRNA and polymerase were significantly blocked by baicalin treatment at early stages of EV71 infection. In addition, baicalin could decrease the expressions of FasL and caspase-3, as well as inhibit the apoptosis of EV71-infected human embryonal rhabdomyosarcoma (RD) cells. Altogether, these results indicate that baicalin exhibits potent antiviral effect on EV71 infection, probably through inhibiting EV71/3D polymerase expression and Fas/FasL signaling pathways.

  • Viruses, Vol. 7, Pages 4734-4755: Regulation of the Wnt/β-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins

  • Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically,β-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/β-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. Viruses 2015, 7 4735 This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/β-catenin pathway.

  • Viruses, Vol. 7, Pages 4707-4733: Regulation of the Host Antiviral State by Intercellular Communications

  • Viruses usually induce a profound remodeling of host cells, including the usurpation of host machinery to support their replication and production of virions to invade new cells. Nonetheless, recognition of viruses by the host often triggers innate immune signaling, preventing viral spread and modulating the function of immune cells. It conventionally occurs through production of antiviral factors and cytokines by infected cells. Virtually all viruses have evolved mechanisms to blunt such responses. Importantly, it is becoming increasingly recognized that infected cells also transmit signals to regulate innate immunity in uninfected neighboring cells. These alternative pathways are notably mediated by vesicular secretion of various virus- and host-derived products (miRNAs, RNAs, and proteins) and non-infectious viral particles. In this review, we focus on these newly-described modes of cell-to-cell communications and their impact on neighboring cell functions. The reception of these signals can have anti- and pro-viral impacts, as well as more complex effects in the host such as oncogenesis and inflammation. Therefore, these“broadcasting” functions, which might be tuned by an arms race involving selective evolution driven by either the host or the virus, constitute novel and original regulations of viral infection, either highly localized or systemic.

  • Viruses, Vol. 7, Pages 4676-4706: Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4

  • Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slenderβ-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem.Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helixis small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix.

  • Viruses, Vol. 7, Pages 4657-4675: Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication

  • Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones) and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ) and RNA interference (siBeclin1) significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.

  • Viruses, Vol. 7, Pages 4640-4656: Flaviviral Replication Complex: Coordination between RNA Synthesis and 5’-RNA Capping

  • Genome replication in flavivirus requires (—) strand RNA synthesis, (+) strand RNA synthesis, and 5’-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (—) RNA synthesis, (+) RNA synthesis, and 5’-RNA capping and methylation.

  • Viruses, Vol. 7, Pages 4624-4639: Wolbachia Do Not Induce Reactive Oxygen Species-Dependent Immune Pathway Activation in Aedes albopictus

  • Aedes albopictus is a major vector of dengue (DENV) and chikungunya (CHIKV) viruses, causing millions of infections annually. It naturally carries, at high frequency, the intracellular inherited bacterial endosymbiont Wolbachia strains wAlbA and wAlbB; transinfection with the higher-density Wolbachia strain wMel from Drosophila melanogaster led to transmission blocking of both arboviruses. The hypothesis that reactive oxygen species (ROS)-induced immune activation plays a role in arbovirus inhibition in this species was examined. In contrast to previous observations in Ae. aegypti, elevation of ROS levels was not observed in either cell lines or mosquito lines carrying the wild-type Wolbachia or higher-density Drosophila Wolbachia strains. There was also no upregulation of genes controlling innate immune pathways or with antioxidant/ROS-producing functions. These data suggest that ROS-mediated immune activation is not an important component of the viral transmission-blocking phenotype in this species.

  • Viruses, Vol. 7, Pages 4602-4623: Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

  • Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages,ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.

  • Viruses, Vol. 7, Pages 4582-4601: The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain

  • Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

  • Viruses, Vol. 7, Pages 4563-4581: eEF1A Interacts with the NS5A Protein and Inhibits the Growth of Classical Swine Fever Virus

  • The NS5A protein of classical swine fever virus (CSFV) is involved in the RNA synthesis and viral replication. However, the NS5A-interacting cellular proteins engaged in the CSFV replication are poorly defined. Using yeast two-hybrid screen, the eukaryotic elongation factor 1A (eEF1A) was identified to be an NS5A-binding partner. The NS5A–eEF1A interaction was confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown and laser confocal microscopy assays. The domain I of eEF1A was shown to be critical for the NS5A–eEF1A interaction. Overexpression of eEF1A suppressed the CSFV growth markedly, and conversely, knockdown of eEF1A enhanced the CSFV replication significantly. Furthermore, eEF1A, as well as NS5A, was found to reduce the translation efficiency of the internal ribosome entry site (IRES) of CSFV in a dose-dependent manner, as demonstrated by luciferase reporter assay. Streptavidin pulldown assay revealed that eEF1A could bind to the CSFV IRES. Collectively, our results suggest that eEF1A interacts with NS5A and negatively regulates the growth of CSFV.

  • Viruses, Vol. 7, Pages 4529-4562: Replication and Inhibitors of Enteroviruses and Parechoviruses

  • The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

  • Viruses, Vol. 7, Pages 4507-4528: Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology

  • DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients.

  • Viruses, Vol. 7, Pages 4488-4506: Genetic Characterization of the Belgian Nephropathogenic Infectious Bronchitis Virus (NIBV) Reference Strain B1648

  • The virulent nephropathogenic infectious bronchitis virus (NIBV) strain B1648 was first isolated in 1984, in Flanders, Belgium. Despite intensive vaccination, B1648 and its variants are still circulating in Europe and North Africa. Here, the full-length genome of this Belgian NIBV reference strain was determined by next generation sequencing (NGS) to understand its evolutionary relationship with other IBV strains, and to identify possible genetic factors that may be associated with the nephropathogenicity. Thirteen open reading frames (ORFs) were predicted in the B1648 strain (51UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-31UTR). ORFs 4b, 4c and 6b, which have been rarely reported in literature, were present in B1648 and most of the other IBV complete genomes. According to phylogenetic analysis of the full-length genome, replicase transcriptase complex, spike protein, partial S1 gene and M protein, B1648 strain clustered with the non-Massachusetts type strains NGA/A116E7/2006, UKr 27-11, QX-like ITA/90254/2005, QX-like CK/SWE/0658946/10, TN20/00, RF-27/99, RF/06/2007 and SLO/266/05. Based on the partial S1 fragment, B1648 clustered with the strains TN20/00, RF-27/99, RF/06/2007 and SLO/266/05 and, further designated as B1648 genotype. The full-length genome of B1648 shared the highest sequence homology with UKr 27-11, Gray, JMK, and NGA/A116E7/2006 (91.2% to 91.6%) and was least related with the reference Beaudette and Massachusetts strains (89.7%). Nucleotide and amino acid sequence analyses indicated that B1648 strain may have played an important role in the evolution of IBV in Europe and North Africa. Further, the nephropathogenicity determinants might be located on the 1a, spike, M and accessory proteins (3a, 3b, 4b, 4c, 5a, 5b and 6b). Overall, strain B1648 is distinct from all the strains reported so far in Europe and other parts of the world.

  • Viruses, Vol. 7, Pages 4482-4487: Special Issue: Gene Therapy with Emphasis on RNA Interference

  • Gene therapy was originally thought to cover replacement of malfunctioning genes in treatment of various diseases. Today, the field has been expanded to application of viral and non-viral vectors for delivery of recombinant proteins for the compensation of missing or insufficient proteins, anti-cancer genes and proteins for destruction of tumor cells, immunostimulatory genes and proteins for stimulation of the host defense system against viral agents and tumors. Recently, the importance of RNA interference and its application in gene therapy has become an attractive alternative for drug development.

  • Viruses, Vol. 7, Pages 4461-4481: Structural and Functional Properties of the Hepatitis C Virus p7 Viroporin

  • The high prevalence of hepatitis C virus (HCV) infection in the human population has triggered intensive research efforts that have led to the development of curative antiviral therapy. Moreover, HCV has become a role model to study fundamental principles that govern the replication cycle of a positive strand RNA virus. In fact, for most HCV proteins high-resolution X-ray and NMR (Nuclear Magnetic Resonance)-based structures have been established and profound insights into their biochemical and biological properties have been gained. One example is p7, a small hydrophobic protein that is dispensable for RNA replication, but crucial for the production and release of infectious HCV particles from infected cells. Owing to its ability to insert into membranes and assemble into homo-oligomeric complexes that function as minimalistic ion channels, HCV p7 is a member of the viroporin family. This review compiles the most recent findings related to the structure and dual pore/ion channel activity of p7 of different HCV genotypes. The alternative conformations and topologies proposed for HCV p7 in its monomeric and oligomeric state are described and discussed in detail. We also summarize the different roles p7 might play in the HCV replication cycle and highlight both the ion channel/pore-like function and the additional roles of p7 unrelated to its channel activity. Finally, we discuss possibilities to utilize viroporin inhibitors for antagonizing p7 ion channel/pore-like activity.

  • Viruses, Vol. 7, Pages 4438-4460: RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms

  • RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies.

  • Viruses, Vol. 7, Pages 4414-4437: Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

  • Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospotr assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)- and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of TH1, TH2, and TH17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN- and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the TH1, TH2, and TH17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.

  • Viruses, Vol. 7, Pages 4385-4413: The Virus-Host Interplay: Biogenesis of +RNA Replication Complexes

  • Positive-strand RNA (+RNA) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. Notable members include West Nile virus, Dengue virus, Chikungunya, Severe acute respiratory syndrome (SARS) Coronavirus and enteroviruses of the Picornaviridae family.Unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +RNA viruses have limited coding capacity and thus rely extensively on host factors for successful infection and propagation. A common feature among these viruses is their ability to dramatically modify cellular membranes to serve as platforms for genome replication and assembly of new virions. These viral replication complexes (VRCs) serve two main functions: To increase replication efficiency by concentrating critical factors and to protect the viral genome from host anti-viral systems. This review summarizes current knowledge of critical host factors recruited to or demonstrated to be involved in the biogenesis and stabilization of +RNA virus VRCs.

  • Viruses, Vol. 7, Pages 4369-4384: Characterization of Humoral Responses Induced by an H7N9 Influenza Virus-Like Particle Vaccine in BALB/C Mice

  • In April 2013, human infections with a novel avian influenza (H7N9) virus emerged in China. It has caused serious concerns for public health throughout the world. However, there is presently no effective treatment, and an A (H7N9) H7 subtype influenza vaccine is not available. Vaccination with virus-like particles (VLPs) has showed considerable promise for many other subtype influenza viruses. To produce H7N9 VLPs, full length, unmodified hemagglutinin (HA), neuraminidase (NA), and matrix1 (M1) genes from the A/Wuxi/1/2013(H7N9) were cloned into a pCDNA5.1 FRT vector. By co-transfection, VLPs containing HA, NA, and M1 were secreted by 293T cells. VLPs were purified by ultracentrifugation and injected into mice by the intramuscular route. In animal experiments, humoral and cellular immunoresponse were all triggered by H7N9 VLPs. High levels of specific antibodies and the isotypes of IgG were detected by ELISA. Anamnestic cellular immune responses were examined by detecting specific cytotoxic T cell for IFN-Υ production in ELISPOT assay. The hemagglutination-inhibition (HAI) against the homologous virus was more than 1:64, and cross-reactive HAI titers against the heterologous virus (H1N1 and H3N2) were more than 1:16. Moreover, VLPs immunized mice showed a rapid increase of neutralizing antibodies,with neutralizing antibody titers more than 1:8, which increased four-fold against PBS immunized mice in week four. By week six, the mice had high neutralization ability against the given strain and held a potent homologous virus neutralizing capacity. Thus, VLPs represent a potential strategy for the development of a safe and effective vaccine against novel avian influenza (H7N9) virus.

  • Viruses, Vol. 7, Pages 4352-4368: Vpu Protein: The Viroporin Encoded by HIV-1

  • Viral protein U (Vpu) is a lentiviral viroporin encoded by human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus (SIV) strains. This small protein of 81 amino acids contains a single transmembrane domain that allows for supramolecular organization via homoligomerization or interaction with other proteins. The topology and trafficking of Vpu through subcellular compartments result in pleiotropic effects in host cells. Notwithstanding the high variability of its amino acid sequence, the functionality of Vpu is well conserved in pandemic virus isolates. This review outlines our current knowledge on the interactions of Vpu with the host cell. The regulation of cellular physiology by Vpu and the validity of this viroporin as a therapeutic target are also discussed.

  • Viruses, Vol. 7, Pages 4326-4351: Translational Control of the HIV Unspliced Genomic RNA

  • Post-transcriptional control in both HIV-1 and HIV-2 is a highly regulated process that commences in the nucleus of the host infected cell and finishes by the expression of viral proteins in the cytoplasm. Expression of the unspliced genomic RNA is particularly controlled at the level of RNA splicing, export, and translation. It appears increasingly obvious that all these steps are interconnected and they result in the building of a viral ribonucleoprotein complex (RNP) that must be efficiently translated in the cytosolic compartment. This review summarizes our knowledge about the genesis, localization, and expression of this viral RNP.

  • Viruses, Vol. 7, Pages 4303-4325: Papillomavirus Infectious Pathways: A Comparison of Systems

  • The HPV viral lifecycle is tightly linked to the host cell differentiation, causing difficulty in growing virions in culture. A system that bypasses the need for differentiating epithelium has allowed for generation of recombinant particles, such as virus-like particles (VLPs), pseudovirions (PsV), and quasivirions (QV). Much of the research looking at the HPV life cycle, infectivity, and structure has been generated utilizing recombinant particles. While recombinant particles have proven to be invaluable, allowing for a rapid progression of the HPV field, there are some significant differences between recombinant particles and native virions and very few comparative studies using native virions to confirm results are done. This review serves to address the conflicting data in the HPV field regarding native virions and recombinant particles.

  • Viruses, Vol. 7, Pages 4282-4302: Whole-Genome Analysis of a Novel Fish Reovirus (MsReV) Discloses Aquareovirus Genomic Structure Relationship with Host in Saline Environments

  • Aquareoviruses are serious pathogens of aquatic animals. Here, genome characterization and functional gene analysis of a novel aquareovirus, largemouth bass Micropterus salmoides reovirus (MsReV), was described. It comprises 11 dsRNA segments (S1–S11) covering 24,024 bp, and encodes 12 putative proteins including the inclusion forming-related protein NS87 and the fusion-associated small transmembrane (FAST) protein NS22. The function of NS22 was confirmed by expression in fish cells. Subsequently, MsReV was compared with two representative aquareoviruses, saltwater fish turbot Scophthalmus maximus reovirus (SMReV) and freshwater fish grass carp reovirus strain 109 (GCReV-109). MsReV NS87 and NS22 genes have the same structure and function with those of SMReV, whereas GCReV-109 is either missing the coiled-coil region in NS79 or thegene-encoding NS22. Significant similarities are also revealed among equivalent genome segments between MsReV and SMReV, but a difference is found between MsReV and GCReV-109. Furthermore, phylogenetic analysis showed that 13 aquareoviruses could be divided into freshwater and saline environments subgroups, and MsReV was closely related to SMReV in saline environments. Consequently, these viruses from hosts in saline environments have more genomic structural similarities than the viruses from hosts in freshwater. This is the first study of the relationships between aquareovirus genomic structure and their host environments.

  • Viruses, Vol. 7, Pages 4254-4281: Public Acceptance of Plant Biotechnology and GM Crops

  • A wide gap exists between the rapid acceptance of genetically modified (GM) crops for cultivation by farmers in many countries and in the global markets for food and feed, and the often-limited acceptance by consumers. This review contrasts the advances of practical applications of agricultural biotechnology with the divergent paths—also affecting the development of virus resistant transgenic crops—of political and regulatory frameworks for GM crops and food in different parts of the world. These have also shaped the different opinions of consumers. Important factors influencing consumer’s attitudes are the perception of risks and benefits, knowledge and trust, and personal values. Recent political and societal developments show a hardening of the negative environment for agricultural biotechnology in Europe, a growing discussion—including calls for labeling of GM food—in the USA, and a careful development in China towards a possible authorization of GM rice that takes the societal discussions into account. New breeding techniques address some consumers’ concerns with transgenic crops, but it is not clear yet how consumers’ attitudes towards them will develop. Discussions about agriculture would be more productive, if they would focus less on technologies, but on common aims and underlying values.

  • Viruses, Vol. 7, Pages 4230-4253: Matrix Metalloproteinase-9 Mediates RSV Infection in Vitro and in Vivo

  • Respiratory Syncytial Virus (RSV) is an important human pathogen associated with substantial morbidity and mortality. The present study tested the hypothesis that RSV infection would increase matrix metalloproteinase (MMP)-9 expression, and that MMP-9 inhibition would decrease RSV replication both in vitro and in vivo. RSV A2 infection of human bronchial epithelial cells increased MMP-9 mRNA and protein release. Cells transfected with siRNA against MMP-9 following RSV infection had lower viral titers. In RSV infected wild-type (WT) mice, MMP-9, airway resistance and viral load peaked at day 2 post infection, and remained elevated on days 4 and 7. RSV infected MMP-9 knockout (KO) mice had decreased lung inflammation. On days 2 and 4 post inoculation, the RSV burden was lower in the MMP-9 KO mice compared to WT controls. In conclusion, our studies demonstrate that RSV infection is a potent stimulus of MMP-9 expression both in vitro and in vivo. Reduction of MMP-9 (via siRNA knockdown, and in MMP-9 KO mice) resulted in decreased viral replication. Our findings suggest MMP-9 is a potential therapeutic target for RSV disease.

  • Viruses, Vol. 7, Pages 4204-4229: Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

  • Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

  • Viruses, Vol. 7, Pages 4186-4203: CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

  • Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

  • Viruses, Vol. 7, Pages 4169-4185: BnSGS3 Has Differential Effects on the Accumulation of CMV, ORMV and TuMV in Oilseed Rape

  • Virus diseases greatly affect oilseed rape (Brassica napus) production. Investigating antiviral genes may lead to the development of disease-resistant varieties of oilseed rape. In this study, we examined the effects of the suppressor of gene silencing 3 in Brassica napus (BnSGS3, a putative antiviral gene) with different genus viruses by constructing BnSGS3-overexpressing (BnSGS3-Ov) and BnSGS3-silenced (BnSGS3-Si) oilseed rape (cv. Zhongshuang No. 6) plants. These three viruses are Oilseed rape mosaic virus (ORMV), Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV). The native BnSGS3 expressed in all examined tissues with the highest expression in siliques. All three viruses induced BnSGS3 expression, but ORMV induced a dramatic increase in the BnSGS3-Ov plants, followed by TuMV and CMV. Upon inoculation with three different viruses, transcript abundance of BnSGS3 gene follows: BnSGS3-Ov aamp;amp;gt; non-transgenic plants aamp;amp;gt; BnSGS3-Si. The accumulation quantities of ORMV and TuMV exhibited a similar trend. However, CMV accumulation showed an opposite trend where virus accumulations were negatively correlated with BnSGS3 expression. The results suggest that BnSGS3 selectively inhibits CMV accumulation but promotes ORMV and TuMV accumulation. BnSGS3 should be used in different ways (up- and down-regulation) for breeding virus-resistant oilseed rape varieties.

  • Viruses, Vol. 7, Pages 4152-4168: Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage

  • Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program.

  • Viruses, Vol. 7, Pages 4131-4151: Pigeon RIG-I Function in Innate Immunity against H9N2 IAV and IBDV

  • Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and mice) and waterfowl RIG-I (ducks and geese) are essential for type I interferon (IFN) synthesis during AIV infection. Like ducks, pigeons are also susceptible to infection but are ineffective propagators and disseminators of AIVs, i.e.,“dead end” hosts for AIVs and even highly pathogenic avian influenza (HPAI). Consequently, we sought to identify pigeon RIG-I and investigate its roles in the detection of A/Chicken/Shandong/ZB/2007 (H9N2) (ZB07), Gansu/Tianshui (IBDV TS) and Beijing/CJ/1980 (IBDV CJ-801) strains in chicken DF-1 fibroblasts or human 293T cells. Pigeon mRNA encoding the putative pigeon RIG-I analogs was identified. The exogenous expression of enhanced green fluorescence protein (EGFP)-tagged pigeon RIG-I and caspase activation and recruitment domains (CARDs), strongly induced antiviral gene (IFN-β, Mx, and PKR) mRNA synthesis, decreased viral gene (M gene and VP2) mRNA expression, and reduced the viral titers of ZB07 and IBDV TS/CJ-801 virus strains in chicken DF-1 cells, but not in 293T cells. We also compared the antiviral abilities of RIG-I proteins from waterfowl (duck and goose) and pigeon. Ourdata indicated that waterfowl RIG-I are more effective in the induction of antiviral genes and the repression of ZB07 and IBDV TS/CJ-801 strain replication than pigeon RIG-I. Furthermore, chicken melanoma differentiation associated gene 5(MDA5)/ mitochondrial antiviral signaling (MAVS) silencing combined with RIG-I transfection suggested that pigeon RIG-I can restore the antiviral response in MDA5-silenced DF-1 cells but not in MAVS-silenced DF-1 cells. In conclusion, these results demonstrated that pigeon RIG-I and CARDs have a strong antiviral ability against AIV H9N2 and IBDV in chicken DF-1 cells but not in human 293T cells.

  • Viruses, Vol. 7, Pages 4119-4130: Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

  • Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previouslyshown to inhibit DENV replication and to interfere with the binding of NS4A(1–48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.

  • Viruses, Vol. 7, Pages 4093-4118: Exosomes: Implications in HIV-1 Pathogenesis

  • Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission.

  • Viruses, Vol. 7, Pages 4075-4092: Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy

  • Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.

  • Viruses, Vol. 7, Pages 4047-4074: The Emerging Role of miRNAs in HTLV-1 Infection and ATLL Pathogenesis

  • Human T-cell leukemia virus (HTLV)-1 is a human retrovirus and the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a fatal malignancy of CD4/CD25+ T lymphocytes. In recent years, cellular as well as virus-encoded microRNA (miRNA) have been shown to deregulate signaling pathways to favor virus life cycle. HTLV-1 does not encode miRNA, but several studies have demonstrated that cellular miRNA expression is affected in infected cells. Distinct mechanisms such as transcriptional, epigenetic or interference with miRNA processing machinery have been involved. This article reviews the current knowledge of the role of cellular microRNAs in virus infection, replication, immune escape and pathogenesis of HTLV-1.

  • Viruses, Vol. 7, Pages 3995-4046: Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design

  • In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with aamp;amp;gt;90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.

  • Viruses, Vol. 7, Pages 3974-3994: Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

  • Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

  • Viruses, Vol. 7, Pages 3954-3973: Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation

  • The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

  • Viruses, Vol. 7, Pages 3937-3953: Tsv-N1: A Novel DNA Algal Virus that Infects Tetraselmis striata

  • Numbering in excess of 10 million per milliliter of water, it is now undisputed that aquatic viruses are one of the major factors shaping the ecology and evolution of Earth’s microbial world. Nonetheless, environmental viral diversity and roles remain poorly understood. Here we report the first thorough characterization of a virus (designated TsV) that infects the coastal marine microalga Tetraselmis striata. Unlike previously known microalgae-infecting viruses, TsV is a small (60 nm) DNA virus, with a 31 kb genome. From a range of eight different strains belonging to the Chlamydomonadaceae family, TsV was only able to infect T. striata. Gene expression dynamics revealed an up-regulation of viral transcripts already 1 h post-infection (p.i.). First clear signs of infection were observed 24 h p.i., with the appearance of viral factories inside the nucleus. TsV assembly was exclusively nuclear. TsV-N1 genome revealed very different from previously known algae viruses (Phycodnaviridae). Putative function and/or homology could be resolved for only 9 of the 33 ORFs encoded. Among those was a surprising DNA polymerase type Delta (only found in Eukaryotes), and two genes with closest homology to genes from human parasites of the urogenital tract. These results support the idea that the diversity of microalgae viruses goes far beyond the Phycodnaviridae family and leave the door open for future studies on implications of microalgae viruses for human health.

  • Viruses, Vol. 7, Pages 3910-3936: Bone Marrow Gene Therapy for HIV/AIDS

  • Bone marrow gene therapy remains an attractive option for treating chronic immunological diseases, including acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV). This technology combines the differentiation and expansion capacity of hematopoietic stem cells (HSCs) with long-term expression of therapeutic transgenes using integrating vectors. In this review we summarize the potential of bone marrow gene therapy for the treatment of HIV/AIDS. A broad range of antiviral strategies are discussed, with a particular focus on RNA-based therapies. The idea is to develop a durable gene therapy that lasts the life span of the infected individual, thus contrasting with daily drug regimens to suppress the virus. Different approaches have been proposed to target either the virus or cellular genes encoding co-factors that support virus replication. Some of these therapies have been tested in clinical trials, providing proof of principle that gene therapy is a safe option for treating HIV/AIDS. In this review several topics are discussed, ranging from the selection of the antiviral molecule and the viral target to the optimal vector system for gene delivery and the setup of appropriate preclinical test systems. The molecular mechanisms used to formulate a cure for HIV infection are described, including the latest antiviral strategies and their therapeutic applications. Finally, a potent combination of anti-HIV genes based on our own research program is described.

  • Viruses, Vol. 7, Pages 3891-3909: Optimization and Characterization of Candidate Strain for Coxsackievirus A16 Inactivated Vaccine

  • Coxsackievirus A16 (CA16) and enterovirus 71 (EV71), both of which can cause hand, foot and mouth disease (HFMD), are responsible for large epidemics in Asian and Pacific areas. Although inactivated EV71 vaccines have completed testing in phase III clinical trials in Mainland China, CA16 vaccines are still under development. A Vero cell-based inactivated CA16 vaccine was developed by our group. Screening identified a CA16 vaccine strain (CC024) isolated from HFMD patients, which had broad cross-protective abilities and satisfied all requirements for vaccine production. Identification of the biological characteristics showed that the CA16CC024 strain had the highest titer (107.5 CCID50/mL) in Vero cells, which would benefit the development of an EV71/CA16 divalent vaccine. A potential vaccine manufacturing process was established, including the selection of optimal time for virus harvesting, membrane for diafiltration and concentration, gel-filtration chromatography for the down-stream virus purification and virus inactivation method. Altogether, the analyses suggested that the CC-16, a limiting dilution clone of the CC024 strain, with good genetic stability, high titer and broad-spectrum immunogenicity, would be the best candidate strain for a CA16 inactivated vaccine. Therefore, our study provides valuable information for the development of a Vero cell-based CA16 or EV71-CA16 divalent inactivated vaccine.

  • Viruses, Vol. 7, Pages 3863-3890: Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia

  • Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted.

  • Viruses, Vol. 7, Pages 3857-3862: A Viral Pilot for HCMV Navigation?

  • gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host.

  • Viruses, Vol. 7, Pages 3835-3856: Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems

  • The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

  • Viruses, Vol. 7, Pages 3816-3834: Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

  • Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

  • Viruses, Vol. 7, Pages 3798-3815: The Apis mellifera Filamentous Virus Genome

  • A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

  • Viruses, Vol. 7, Pages 3789-3797: Learning from Ebola Virus: How to Prevent Future Epidemics

  • The recent Ebola virus disease (EVD) epidemic in Guinea, Liberia and Sierra Leone demonstrated that the World Health Organization (WHO) is incapable to control outbreaks of infectious diseases in less developed regions of the world. This essay analyses the causes for the failure of the international response and proposes four measures to improve resilience, early detection and response to future outbreaks of infectious diseases.

  • Viruses, Vol. 7, Pages 3768-3788: Cloned Defective Interfering Influenza RNA and a Possible Pan-Specific Treatment of Respiratory Virus Diseases

  • Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases in vivo using as an example a highly active cloned influenza A DI genome and virus that protects broadly in preclinical trials against different subtypes of influenza A and against non-influenza A respiratory viruses. This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials.

  • Viruses, Vol. 7, Pages 3741-3767: Tissue Barriers to Arbovirus Infection in Mosquitoes

  • Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts.

  • Viruses, Vol. 7, Pages 3719-3740: Adenovirus 36 and Obesity: An Overview

  • There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed.

  • Viruses, Vol. 7, Pages 3703-3718: Resistance against Integrase Strand Transfer Inhibitors and Relevance to HIV Persistence

  • Drug resistance prevents the successful treatment of HIV-positive individuals by decreasing viral sensitivity to a drug or a class of drugs. In addition to transmitted resistant viruses, treatment-naïve individuals can be confronted with the problem of drug resistance through de novo emergence of such variants. Resistant viruses have been reported for every antiretroviral drug tested so far, including the integrase strand transfer inhibitors raltegravir, elvitegravir and dolutegravir. However, de novo resistant variants against dolutegravir have been found in treatment-experienced but not in treatment-naïve individuals, a characteristic that is unique amongst antiretroviral drugs. We review here the issue of drug resistance against integrase strand transfer inhibitors as well as both pre-clinical and clinical studies that have led to the identification of the R263K mutation in integrase as a signature resistance substitution for dolutegravir. We also discuss how the topic of drug resistance against integrase strand transfer inhibitors may have relevance in regard to the nature ofthe HIV reservoir and possible HIV curative strategies.

  • Viruses, Vol. 7, Pages 3675-3702: Resistance to Rhabdoviridae Infection and Subversion of Antiviral Responses

  • Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play a role in mediating the antiviral response in vivo using gene knockout mouse models. IFNs inhibit rhabdovirus replication at different stages via the induction of a variety of ISGs. This review will discuss how individual ISG products confer resistance to rhabdoviruses by blocking viral entry, degrading single stranded viral RNA, inhibiting viral translation or preventing release of virions from the cell. Furthermore, this review will highlight how these viruses counteract the host IFN system.

  • Viruses, Vol. 7, Pages 3647-3674: Early Events in Chikungunya Virus Infection—From Virus CellBinding to Membrane Fusion

  • Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research.

  • Viruses, Vol. 7, Pages 3625-3646: Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

  • Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  • Viruses, Vol. 7, Pages 3603-3624: Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

  • Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans.

  • Viruses, Vol. 7, Pages 3586-3602: Genome Characterization, Prevalence and Distribution of a Macula-Like Virus from Apis mellifera and Varroa destructor

  • Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).

  • Viruses, Vol. 7, Pages 3574-3585: Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses

  • All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  • Viruses, Vol. 7, Pages 3552-3573: Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis

  • Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.

  • Viruses, Vol. 7, Pages 3530-3551: The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy

  • Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

  • Viruses, Vol. 7, Pages 3506-3529: Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters

  • Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

  • Viruses, Vol. 7, Pages 3500-3505: Less Grease, Please. Phosphatidylethanolamine Is the Only Lipid Required for Replication of a (+)RNA Virus

  • All positive strand RNA viruses of eukaryotes replicate their genomes in association with membranes. These viruses actively change cellular lipid metabolism to build replication membranes enriched in specific lipids. The ubiquitous use of membranes by positive strand RNA viruses apparently holds major evolutionary advantages; however our understanding of the mechanistic role of membranes, let alone of specific lipid components of the membrane bilayer, in the viral replication cycle is minimal. The replication complexes that can be isolated from infected cells, or reconstituted in vitro from crude cell lysates, do not allow controlled manipulation of the membrane constituents thus limiting their usefulness for understanding how exactly membranes support the replication reaction. Recent work from Peter Nagy group demonstrates that replication of a model positive strand RNA virus can be reconstituted in the in vitro reaction with liposomes of chemically defined composition and reveals an exclusive role of phosphatidylethanolamine in sustaining efficient viral RNA replication. This study opens new possibilities for investigation of membrane contribution in the replication process that may ultimately lead to development of novel broad spectrum antiviral compounds targeting the membrane-dependent elements of the replication cycle conserved among diverse groups of viruses.

  • Viruses, Vol. 7, Pages 3483-3499: Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses

  • Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR) and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%–99% similar, whereas Kroon virus had a low similarity (90%–91%) with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment.

  • Viruses, Vol. 7, Pages 3462-3482: Viroporins, Examples of the Two-Stage Membrane Protein Folding Model

  • Viroporins are small,α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.

  • Viruses, Vol. 7, Pages 3443-3461: The Subcellular Localisation of the Human Papillomavirus (HPV) 16 E7 Protein in Cervical Cancer Cells and Its Perturbation by RNA Aptamers

  • Human papillomavirus (HPV) is the most common viral infection of the reproductive tract, affecting both men and women. High-risk oncogenic types are responsible for almost 90% of anogenital and oropharyngeal cancers including cervical cancer. Some of the HPV“early” genes, particularly E6 and E7, are known to act as oncogenes that promote tumour growth and malignant transformation. Most notably, HPV-16 E7 interacts with the tumour suppressor protein pRb, promoting its degradation, leading to cell cycle dysregulation in infected cells. We have previously shown that an RNA aptamer (termed A2) selectively binds to HPV16 E7 and is able to induce apoptosis in HPV16-transformed cervical carcinoma cell lines (SiHa) through reduction of E7 levels. In this study, we investigated the effects of the A2 aptamer on E7 localisation in order to define its effects on E7 activity. We demonstrate for the first time that E7 localised to the plasma membrane. In addition, we show that A2 enhanced E7 localisation in the ER and that the A2-mediated reduction of E7 was not associated with proteasomal degradation. These data suggest that A2 perturbs normal E7 trafficking through promoting E7 ER retention.

  • Viruses, Vol. 7, Pages 3420-3442: Experimental Inoculation of Egyptian Rousette Bats (Rousettus aegyptiacus) with Viruses of the Ebolavirus and Marburgvirus Genera

  • The Egyptian rousette bat (Rousettus aegyptiacus) is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston), and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus—single reservoir host relationship.

  • Viruses, Vol. 7, Pages 3392-3419: Plant Translation Factors and Virus Resistance

  • Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable.

  • Viruses, Vol. 7, Pages 3380-3391: NLRP3 Inflammasome Activation by Viroporins of Animal Viruses

  • Viroporins are a group of low-molecular-weight proteins containing about 50–120 amino acid residues, which are encoded by animal viruses. Viroporins are involved in several stages of the viral life cycle, including viral gene replication and assembly, as well as viral particle entry and release. Viroporins also play an important role in the regulation of antiviral innate immune responses, especially in inflammasome formation and activation, to ensure the completion of the viral life cycle. By reviewing the research progress made in recent years on the regulation of the NLRP3 inflammasome by viroporins of animal viruses, we aim to understand the importance of viroporins in viral infection and to provide a reference for further research and development of novel antiviral drugs.

  • Viruses, Vol. 7, Pages 3361-3379: Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

  • Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detailφPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

  • Viruses, Vol. 7, Pages 3345-3360: RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

  • An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  • Viruses, Vol. 7, Pages 3329-3344: Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry

  • Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.

  • Viruses, Vol. 7, Pages 3310-3328: Phylodynamics of H5N1 Highly Pathogenic Avian Influenza in Europe, 2005–2010: Potential for Molecular Surveillance of New Outbreaks

  • Previous Bayesian phylogeographic studies of H5N1 highly pathogenic avian influenza viruses (HPAIVs) explored the origin and spread of the epidemic from China into Russia, indicating that HPAIV circulated in Russia prior to its detection there in 2005. In this study, we extend this research to explore the evolution and spread of HPAIV within Europe during the 2005–2010 epidemic, using all available sequences of the hemagglutinin (HA) and neuraminidase (NA) gene regions that were collected in Europe and Russia during the outbreak. We use discrete-trait phylodynamic models within a Bayesian statistical framework to explore the evolution of HPAIV. Our results indicate that the genetic diversity and effective population size of HPAIV peaked between mid-2005 and early 2006, followed by drastic decline in 2007, which coincides with the end of the epidemic in Europe. Our results also suggest that domestic birds were the most likely source of the spread of the virus from Russia into Europe. Additionally, estimates of viral dispersal routes indicate that Russia, Romania, and Germany were key epicenters of these outbreaks. Our study quantifies the dynamics of a major European HPAIV pandemic and substantiates the ability of phylodynamic models to improvemolecular surveillance of novel AIVs.

  • Viruses, Vol. 7, Pages 3285-3309: Honey Bee Infecting Lake Sinai Viruses

  • Honey bees are critical pollinators of important agricultural crops. Recently, high annual losses of honey bee colonies have prompted further investigation of honey bee infecting viruses. To better characterize the recently discovered and very prevalent Lake Sinai virus (LSV) group, we sequenced currently circulating LSVs, performed phylogenetic analysis, and obtained images of LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, the first detection of LSV4 in the US, and the discovery of LSV6 and LSV7. We detected LSV1 and LSV2 in the Varroa destructor mite, and determined that a large proportion of LSV2 is found in the honey bee gut, suggesting that vector-mediated, food-associated, and/or fecal-oral routes may be important for LSV dissemination. Pathogen-specific quantitative PCR data, obtained from samples collected during a small-scale monitoring project, revealed that LSV2, LSV1, Black queen cell virus (BQCV), and Nosema ceranae were more abundant in weak colonies than strong colonies within this sample cohort. Together, these results enhance our current understanding of LSVs and illustrate the importance of future studies aimed at investigating the role of LSVs and other pathogens on honey bee health at both the individual and colony levels.
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