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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

  • Current Issue
  • Current Issue of Viruses

    Viruses

  • 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.

  • Viruses, Vol. 7, Pages 3261-3284: Viral Membrane Channels: Role and Function in the Virus Life Cycle

  • Viroporins are small, hydrophobic trans-membrane viral proteins that oligomerize to form hydrophilic pores in the host cell membranes. These proteins are crucial for the pathogenicity and replication of viruses as they aid in various stages of the viral life cycle, from genome uncoating to viral release. In addition, the ion channel activity of viroporin causes disruption in the cellular ion homeostasis, in particular the calcium ion. Fluctuation in the calcium level triggers the activation of the host defensive programmed cell death pathways as well as the inflammasome, which in turn are being subverted for the viruses’ replication benefits. This review article summarizes recent developments in the functional investigation of viroporins from various viruses and their contributions to viral replication and virulence.

  • Viruses, Vol. 7, Pages 3241-3260: Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

  • Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors.

  • Viruses, Vol. 7, Pages 3226-3240: RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

  • Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.

  • Viruses, Vol. 7, Pages 3204-3225: Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses

  • Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.

  • Viruses, Vol. 7, Pages 3201-3203: Correction: Hollingworth, R.; Grand, R.J. Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses. Viruses 2015, 7, 2542-2591

  • We have noted a number of errors in the references of this manuscript.[...]

  • Viruses, Vol. 7, Pages 3186-3200: Coordinated DNA Replication by the Bacteriophage T4 Replisome

  • The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.

  • Viruses, Vol. 7, Pages 3172-3185: The Effect of Oral Administration of dsRNA on Viral Replication and Mortality in Bombus terrestris

  • Israeli acute paralysis virus (IAPV), a single-stranded RNA virus, has a worldwide distribution and affects honeybees as well as other important pollinators. IAPV infection in honeybees has been successfully repressed by exploiting the RNA interference (RNAi) pathway of the insect’s innate immune response with virus-specific double stranded RNA (dsRNA). Here we investigated the effect of IAPV infection in the bumblebee Bombus terrestris and its tissue tropism. B. terrestris is a common pollinator of wild flowers in Europe and is used for biological pollination in agriculture. Infection experiments demonstrated a similar pathology and tissue tropism in bumblebees as reported for honeybees. The effect of oral administration of virus-specific dsRNA was examined and resulted in an effective silencing of the virus, irrespective of the length. Interestingly, we observed that non-specific dsRNA was also efficient against IAPV. However further study is needed to clarify the precise mechanism behind this effect. Finally we believe that our data are indicative of the possibility to use dsRNA for a broad range viral protection in bumblebees.

  • Viruses, Vol. 7, Pages 3155-3171: Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease

  • Enterovirus A71 (EV-A71) in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/β receptor 1 (IFNAR1) to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-β Anti-EV-A71 activities of CW-33 alone and in combination with IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM). Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2',5'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

  • Viruses, Vol. 7, Pages 3130-3154: Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays

  • Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes.

  • Viruses, Vol. 7, Pages 3116-3129: Important Role of the IL-32 Inflammatory Network in the Host Response against Viral Infection

  • The pro-inflammatory cytokine interleukin (IL)-32 has gained much attention recently because of its important role in the inflammatory network. Since the discovery of IL-32 in 2005, our appreciation for its diverse roles continues to grow. Recent studies have discovered the antiviral effects induced by IL-32 and its associated regulatory mechanisms. The interactions between IL-32 and various cytokines including cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), interferon (IFN)-λ1, interleukin (IL)-6, and soluble IL-6 receptor have been described. This review aims to integrate these new findings into explicit concepts and raises the intriguing possibility of IL-32 as a therapeutic target.

  • Viruses, Vol. 7, Pages 3076-3115: Overview on Sobemoviruses and a Proposal for the Creation of the Family Sobemoviridae

  • The genus Sobemovirus, unassigned to any family, consists of viruses with single-stranded plus-oriented single-component RNA genomes and small icosahedral particles. Currently, 14 species within the genus have been recognized by the International Committee on Taxonomy of Viruses (ICTV) but several new species are to be recognized in the near future. Sobemovirus genomes are compact with a conserved structure of open reading frames and with short untranslated regions. Several sobemoviruses are important pathogens. Moreover, over the last decade sobemoviruses have become important model systems to study plant virus evolution. In the current review we give an overview of the structure and expression of sobemovirus genomes, processing and functions of individual proteins, particle structure, pathology and phylogenesis of sobemoviruses as well as of satellite RNAs present together with these viruses. Based on a phylogenetic analysis we propose that a new family Sobemoviridae should be recognized including the genera Sobemovirus and Polemovirus. Finally, we outline the future perspectives and needs for the research focusing on sobemoviruses.

  • Viruses, Vol. 7, Pages 3053-3075: HIV Rev Assembly on the Rev Response Element (RRE): A Structural Perspective

  • HIV-1 Rev is an ~13 kD accessory protein expressed during the early stage of virus replication. After translation, Rev enters the nucleus and binds the Rev response element (RRE), a ~350 nucleotide, highly structured element embedded in the env gene in unspliced and singly spliced viral RNA transcripts. Rev-RNA assemblies subsequently recruit Crm1 and other cellular proteins to form larger complexes that are exported from the nucleus. Once in the cytoplasm, the complexes dissociate and unspliced and singly-spliced viral RNAs are packaged into nascent virions or translated into viral structural proteins and enzymes, respectively. Rev binding to the RRE is a complex process, as multiple copies of the protein assemble on the RNA in a coordinated fashion via a series of Rev-Rev and Rev-RNA interactions. Our understanding of the nature of these interactions has been greatly advanced by recent studies using X-ray crystallography, small angle X-ray scattering (SAXS) and single particle electron microscopy as well as biochemical and genetic methodologies. These advances are discussed in detail in this review, along with perspectives on development of antiviral therapies targeting the HIV-1 RRE.

  • Viruses, Vol. 7, Pages 3035-3052: IFITMs from Mycobacteria Confer Resistance to Influenza Virus When Expressed in Human Cells

  • Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.

  • Viruses, Vol. 7, Pages 3019-3034: A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

  • Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophageɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

  • Viruses, Vol. 7, Pages 2999-3018: APOBEC3 Interference during Replication of Viral Genomes

  • Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s) is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  • Viruses, Vol. 7, Pages 2980-2998: Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma

  • Oncolytic Newcastle disease virus (NDV) might be a promising new therapeutic agent for the treatment of pancreatic cancer. We evaluated recombinant NDVs (rNDVs) expressing interferon (rNDV-hIFNβ-F\(_{\rm{0}}\)) or an IFN antagonistic protein (rNDV-NS1-F\(_{\rm{0}}\)), as well as rNDV with increased virulence (rNDV-F\(_{\rm{3aa}}\)) for oncolytic efficacy in human pancreatic adenocarcinoma cells. Expression of additional proteins did not hamper virus replication or cytotoxic effects on itself. However, expression of interferon, but not NS1, resulted in loss of multicycle replication. Conversely, increasing the virulence (rNDV-F\(_{\rm{3aa}}\)) resulted in enhanced replication of the virus. Type I interferon was produced in high amounts by all tumor cells inoculated with rNDV-hIFNβ -F\(_{\rm{0}}\), while inoculation with rNDV-NS1-F\(_{\rm{0}}\) resulted in a complete block of interferon production in most cells. Inoculation of human pancreatic adenocarcinoma cells with rNDV-F\(_{\rm{3aa}}\) caused markedly more cytotoxicity compared to rNDV-F\(_{\rm{0}}\), while inoculation with rNDV-hIFNβ -F\(_{\rm{0}}\) and rNDV-NS1-F\(_{\rm{0}}\) induced cytotoxic effects comparable to those induced by the parental rNDV-F\(_{\rm{0}}\). Evaluation \textit{in vivo} using mice bearing subcutaneous pancreatic cancer xenografts revealed that only intratumoral injection with rNDV-F\(_{\rm{3aa}}\) resulted in regression of tumors. We conclude that although lentogenic rNDVs harboring proteins that modulate the type I interferon pathway proteins do have an oncolytic effect, a more virulent mesogenic rNDV might be needed to improve oncolytic efficacy.

  • Viruses, Vol. 7, Pages 2965-2979: Lentiviral Vector Mediated Claudin1 Silencing Inhibits Epithelial to Mesenchymal Transition in Breast Cancer Cells

  • Breast cancer has a high incidence and mortality rate worldwide. Several viral vectors including lentiviral, adenoviral and adeno-associated viral vectors have been used in gene therapy for various forms of human cancer, and have shown promising effects in controlling tumor development. Claudin1 (CLDN1) is a member of the tetraspan transmembrane protein family that plays a major role in tight junctions and is associated with tumor metastasis. However, the role of CLDN1 in breast cancer is largely unexplored. In this study, we tested the therapeutic potential of silencing CLDN1 expression in two breast cancer (MDA-MB-231 and MCF7) cell lines using lentiviral vector mediated RNA interference. We found that a CLDN1 short hairpin (shRNA) construct efficiently silenced CLDN1 expression in both breast cancer cell lines, and CLDN1 knockdown resulted in reduced cell proliferation, survival, migration and invasion. Furthermore, silencing CLDN1 inhibited epithelial to mesenchymal transition (EMT) by upregulating the epithelial cell marker, E-cadherin, and downregulating mesenchymal markers, smooth muscle cell alpha-actin (SMA) and Snai2. Our data demonstrated that lentiviral vector mediated CLDN1 RNA interference has great potential in breast cancer gene therapy by inhibiting EMT and controlling tumor cell growth.

  • Viruses, Vol. 7, Pages 2943-2964: The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

  • Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation productof the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigatingthe structure and function of the CHIKV CP.

  • Viruses, Vol. 7, Pages 2928-2942: The Roles of Syncytin-Like Proteins in Ruminant Placentation

  • Recent developments in genome sequencing techniques have led to the identification of huge numbers of endogenous retroviruses (ERV) in various mammals. ERVs, which occupy 8%–13% of mammalian genomes, are believed to affect mammalian evolution and biological diversity. Although the functional significance of most ERVs remains to be elucidated, several ERVs are thought to have pivotal roles in host physiology. We and other groups recently identified ERV envelope proteins (e.g., Fematrin-1, Syncytin-Rum1, endogenous Jaagsiekte sheep retrovirus Env) that may determine the morphogenesis of the unique fused trophoblast cells, termed trinucleate cells and syncytial plaques, found in ruminant placentas; however, there are still a number of outstanding issues with regard to the role of ERVs that remain to be resolved. Here, we review what is known about how these ERVs have contributed to the development of ruminant-specific trophoblast cells.

  • Viruses, Vol. 7, Pages 2908-2927: Activation of DNA Damage Response Pathways during Lytic Replication of KSHV

  • Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

  • Viruses, Vol. 7, Pages 2884-2907: Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

  • Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.

  • Viruses, Vol. 7, Pages 2858-2883: Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

  • Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i) the envelope protein in coronaviruses and (ii) the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  • Viruses, Vol. 7, Pages 2834-2857: The Emerging Roles of Viroporins in ER Stress Response and Autophagy Induction during Virus Infection

  • Viroporins are small hydrophobic viral proteins that oligomerize to form aqueous pores on cellular membranes. Studies in recent years have demonstrated that viroporins serve important functions during virus replication and contribute to viral pathogenicity. A number of viroporins have also been shown to localize to the endoplasmic reticulum (ER) and/or its associated membranous organelles. In fact, replication of most RNA viruses is closely linked to the ER, and has been found to cause ER stress in the infected cells. On the other hand, autophagy is an evolutionarily conserved“self-eating” mechanism that is also observed in cells infected with RNA viruses. Both ER stress and autophagy are also known to modulate a wide variety of signaling pathways including pro-inflammatory and innate immune response, thereby constituting a major aspect of host-virus interactions. In this review, the potential involvement of viroporins in virus-induced ER stress and autophagy will be discussed.

  • Viruses, Vol. 7, Pages 2816-2833: Hydrogen Peroxide Induce Human Cytomegalovirus Replication through the Activation of p38-MAPK Signaling Pathway

  • Human cytomegalovirus (HCMV) is a major risk factor in transplantation and AIDS patients, which induces high morbidity and mortality. These patients infected with HCMV experience an imbalance of redox homeostasis that cause accumulation of reactive oxygen species (ROS) at the cellular level. H2O2, the most common reactive oxygen species, is the main byproduct of oxidative metabolism. However, the function of H2O2 on HCMV infection is not yet fully understood and the effect and mechanism of N-acetylcysteine (NAC) on H2O2-stimulated HCMV replication is unclear. We, therefore, examined the effect of NAC on H2O2-induced HCMV production in human foreskin fibroblast cells. In the present study, we found that H2O2 enhanced HCMV lytic replication through promoting major immediate early (MIE) promoter activity and immediate early (IE) gene transcription. Conversely, NAC inhibited H2O2-upregulated viral IE gene expression and viral replication. The suppressive effect of NAC on CMV in an acute CMV-infected mouse model also showed a relationship between antioxidants and viral lytic replication. Intriguingly, the enhancement of HCMV replication via supplementation with H2O2 was accompanied with the activation of the p38 mitogen-activated protein kinase pathway. Similar to NAC, the p38 inhibitor SB203580 inhibited H2O2-induced p38 phosphorylation and HCMV upregulation, while upregulation of inducible ROS was unaffected. These results directly relate HCMV replication to H2O2, suggesting that treatment with antioxidants may be an attractive preventive and therapeutic strategy for HCMV.

  • Viruses, Vol. 7, Pages 2794-2815: Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis

  • During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization.

  • Viruses, Vol. 7, Pages 2771-2793: The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

  • Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 andɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\(_{\rm{R}}\)3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\(_{\rm{R}}\)3) upstream of the prophage repressor gene. The O\(_{\rm{R}}\)3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

  • Viruses, Vol. 7, Pages 2745-2770: HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

  • Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter,leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

  • Viruses, Vol. 7, Pages 2727-2744: Characterization of Novel Transcripts in Pseudorabies Virus

  • In this study we identified two 3′-coterminal RNA molecules in the pseudorabies virus. The highly abundant short transcript (CTO-S) proved to be encoded between the ul21 and ul22 genes in close vicinity of the replication origin (OriL) of the virus. The less abundant long RNA molecule (CTO-L) is a transcriptional readthrough product of the ul21 gene and overlaps OriL. These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system. It emerged that transcription of the CTOs is fully dependent on the viral transactivator protein IE180 and CTO-S is not a microRNA precursor. We propose an interaction between the transcription and replication machineries at this genomic location, which might play an important role in the regulation of DNA synthesis.

  • Viruses, Vol. 7, Pages 2704-2726: Hepatitis E Virus Genotype 3 Diversity: Phylogenetic Analysis and Presence of Subtype 3b in Wild Boar in Europe

  • An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe.

  • Viruses, Vol. 7, Pages 2683-2703: The Use of Convalescent Sera in Immune-Electron Microscopy to Detect Non-Suspected/New Viral Agents

  • Negative staining electron microscopy methods can be employed for the diagnosis of viral particles in animal samples. In fact, negative staining electron microscopy methods are used to identify viruses, especially in minor species and wild animals, when no other methods are available and in cases of rare, emerging or re-emerging infections. In particular, immune-electron-microscopy with convalescent sera is employed to detect etiological agents when there are undiagnosed clinical outbreaks, when alternative diagnostic methods fail due to the lack of immunological reagents and primers, and when there is no indicative clinical suspect. An overview of immune-electron-microscopy with convalescent sera’s use in the diagnosis of new and unsuspected viruses in animals of domestic and wild species is provided through the descriptions of the following four diagnostic veterinary cases: (I) enteric viruses of pigs: Porcine Rotavirus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus and Porcine Torovirus; (II) Rotavirus and astrovirus in young turkeys with enteritis; (III) Parvovirus-like particles in pheasants; and (IV) Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus.

  • Viruses, Vol. 7, Pages 2668-2682: Nucleocytoplasmic Shuttling of Influenza A Virus Proteins

  • Influenza viruses transcribe and replicate their genomes in the nuclei of infected host cells. The viral ribonucleoprotein (vRNP) complex of influenza virus is the essential genetic unit of the virus. The viral proteins play important roles in multiple processes, including virus structural maintenance, mediating nucleocytoplasmic shuttling of the vRNP complex, virus particle assembly, and budding. Nucleocytoplasmic shuttling of viral proteins occurs throughout the entire virus life cycle. This review mainly focuses on matrix protein (M1), nucleoprotein (NP), nonstructural protein (NS1), and nuclear export protein (NEP), summarizing the mechanisms of their nucleocytoplasmic shuttling and the regulation of virus replication through their phosphorylation to further understand the regulation of nucleocytoplasmic shuttling in host adaptation of the viruses.

  • Viruses, Vol. 7, Pages 2654-2667: Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites

  • Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

  • Viruses, Vol. 7, Pages 2641-2653: The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

  • Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.

  • Viruses, Vol. 7, Pages 2618-2640: Insights into Human Astrocyte Response to H5N1 Infection by Microarray Analysis

  • Influenza virus infects not only the respiratory system but also the central nervous system (CNS), leading to influenza-associated encephalopathy and encephalitis. Astrocytes are essential for brain homeostasis and neuronal function. These cells can also be infected by influenza virus. However, genome-wide changes in response to influenza viral infection in astrocytes have not been defined. In this study, we performed gene profiling of human astrocytes in response to H5N1. Innate immune and pro-inflammatory responses were strongly activated at 24 h post-infection (hpi). Antiviral genes, as well as several cytokines and chemokines, including CXCL9, CXCL10, and CXCL11, were robustly induced. Phosphorylation of p65 and p38 can be activated by viral infection, suggesting their potential critical roles in H5N1-induced pro-inflammatory response. Moreover, H5N1 infection significantly upregulated the gene expressions related to the neuroactive ligand-receptor interaction pathway at 24 hpi, such as MC2R, CHRNG, P2RY13, GABRA1, and HRH2, which participant in synaptic transmission and may take part in CNS disorders induced by H5N1 infection. Targeting key components of innate immune response and the neuroactive ligand-receptor interaction pathway may provide a strategy to control H5N1-induced encephalopathy and encephalitis. This research can contribute to the understanding of H5N1 pathogenesis in astrocytes.

  • Viruses, Vol. 7, Pages 2592-2617: Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

  • Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viralentry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  • Viruses, Vol. 7, Pages 2542-2591: Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses

  • With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers.

  • Viruses, Vol. 7, Pages 2534-2541: A Taxonomic Review of Clostridium difficile Phages and Proposal of a Novel Genus, “Phimmp04likevirus”

  • Currently, only three phages that infect the medically important bacterium Clostridium difficile have been discussed by the International Committee of Viral Taxonomy (ICTV). They are all myoviruses, and have been assigned to the genus“phicd119likevirus”. An additional nine phages have since been described in the literature with their genome data available. The Phicd119likevirus is named after the type species: the myovirus ΦCD119 which was the first C. difficile phage to be sequenced. The two additional myoviruses, ϕCD27 and φC2, also fall into this genus based on the similarity of their genome and morphological characteristics. The other nine phages have not been assigned to this genus, and four of them do not fit the criteria for the current taxonomic grouping. We have applied protein clustering analysis to determine their phylogenetic relationships. From these results we propose an additional myoviridae genus, that we term “phiMMP04likevirus”.

  • Viruses, Vol. 7, Pages 2518-2533: Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus

  • The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.

  • Viruses, Vol. 7, Pages 2507-2517: IPNV Antigen Uptake and Distribution in Atlantic Salmon Following Oral Administration

  • One impediment to the successful oral vaccination in fish is the hostile stomach environment that antigens must cross. Furthermore, uptake of antigens from the gut to systemic distribution is required for induction of systemic immunity, the dynamics of which are poorly understood. In the present study, groups of Atlantic salmon parr were intubated with live or inactivated infectious pancreatic necrosis virus (IPNV), either orally or anally. At 1, 24 and 72 h post infection (p.i.), the fish were sacrificed. Serum was used for assessing IPNV by ELISA, while formalin-fixed head-kidney, spleen, liver and intestine tissues were used for the demonstration of antigens by immunohistochemistry. Both live and inactivated IPNV antigens were observed in enterocytes of the intestines and in immune cells of the head-kidneys and spleens of all groups. In the liver, no antigens were observed in any of the groups. Significantly higher serum antigen OD values (p aamp;amp;lt; 0.04) were observed in orally- compared to anally-intubated fish. By contrast, no difference (p = 0.05) was observed in tissue antigens between these groups by immunohistochemistry. No significant difference (p = 0.05) in serum antigens was observed between groups intubated with live and inactivated IPNV, while in tissues, significantly more antigens (p aamp;amp;lt; 0.03) were observe in the latter compared to the former. These findings demonstrate that both live and inactivated IPNV are taken up by enterocytes in the intestines of Atlantic salmon, likely by receptor-mediated mechanisms. Higher IPNV uptake by the oral compared to anal route suggests that both the anterior and posterior intestines are important for the uptake of the virus and that IPNV is resistant to gastric degradation of the Atlantic salmon stomach.

  • Viruses, Vol. 7, Pages 2485-2506: High-Risk Human Papillomavirus Targets Crossroads in Immune Signaling

  • Persistent infections with a high-risk type human papillomavirus (hrHPV) can progress to cancer. High-risk HPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years despite the fact that KCs are well equipped to detect and initiate immune responses to invading pathogens. Viral persistence is achieved by active interference with KCs innate and adaptive immune mechanisms. To this end hrHPV utilizes proteins encoded by its viral genome, as well as exploits cellular proteins to interfere with signaling of innate and adaptive immune pathways. This results in impairment of interferon and pro-inflammatory cytokine production and subsequent immune cell attraction, as well as resistance to incoming signals from the immune system. Furthermore, hrHPV avoids the killing of infected cells by interfering with antigen presentation to antigen-specific cytotoxic T lymphocytes. Thus, hrHPV has evolved multiple mechanisms to avoid detection and clearance by both the innate and adaptive immune system, the molecular mechanisms of which will be dealt with in detail in this review.

  • Viruses, Vol. 7, Pages 2470-2484: Molecular Characterization of a Novel Positive-Sense, Single-Stranded RNA Mycovirus Infecting the Plant Pathogenic Fungus Sclerotinia sclerotiorum

  • Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5'- and 3'-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.

  • Viruses, Vol. 7, Pages 2450-2469: The Role of the DNA Damage Response throughout the Papillomavirus Life Cycle

  • The DNA damage response (DDR) maintains genomic integrity through an elaborate network of signaling pathways that sense DNA damage and recruit effector factors to repair damaged DNA. DDR signaling pathways are usurped and manipulated by the replication programs of many viruses. Here, we review the papillomavirus (PV) life cycle, highlighting current knowledge of how PVs recruit and engage the DDR to facilitate productive infection.

  • Viruses, Vol. 7, Pages 2428-2449: Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression

  • The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation.

  • Viruses, Vol. 7, Pages 2404-2427: A Novel Iminosugar UV-12 with Activity against the Diverse Viruses Influenza and Dengue (Novel Iminosugar Antiviral for Influenza and Dengue)

  • Iminosugars are capable of targeting the life cycles of multiple viruses by blocking host endoplasmic reticulumα-glucosidase enzymes that are required for competent replication of a variety of enveloped, glycosylated viruses. Iminosugars as a class are approved for use in humans with diseases such as diabetes and Gaucher’s disease, providing evidence for safety of this class of compounds. The in vitro antiviral activity of iminosugars has been described in several publications with a subset of these demonstrating in vivo activity against flaviviruses, herpesviruses, retroviruses and filoviruses. Although there is compelling non-clinical in vivo evidence of antiviral efficacy, the efficacy of iminosugars as antivirals has yet to be demonstrated in humans. In the current study, we report a novel iminosugar, UV-12, which has efficacy against dengue and influenza in mouse models. UV-12 exhibits drug-like properties including oral bioavailability and good safety profile in mice and guinea pigs. UV-12 is an example of an iminosugar with activity against multiple virus families that should be investigated in further safety and efficacy studies and demonstrates potential value of this drug class as antiviral therapeutics.

  • Viruses, Vol. 7, Pages 2378-2403: Influence of Cellular Trafficking Pathway on Bluetongue Virus Infection in Ovine Cells

  • Bluetongue virus (BTV), a non-enveloped arbovirus, causes hemorrhagic disease in ruminants. However, the influence of natural host cell proteins on BTV replication process is not defined. In addition to cell lysis, BTV also exits non-ovine cultured cells by non-lytic pathways mediated by nonstructural protein NS3 that interacts with virus capsid and cellular proteins belonging to calpactin and ESCRT family. The PPXY late domain motif known to recruit NEDD4 family of HECT ubiquitin E3 ligases is also highly conserved in NS3. In this study using a mixture of molecular, biochemical and microscopic techniques we have analyzed the importance of ovine cellular proteins and vesicles in BTV infection. Electron microscopic analysis of BTV infected ovine cells demonstrated close association of mature particles with intracellular vesicles. Inhibition of Multi Vesicular Body (MVB) resident lipid phosphatidylinositol-3-phosphate resulted in decreased total virus titre suggesting that the vesicles might be MVBs. Proteasome mediated inhibition of ubiquitin or modification of virus lacking the PPXY in NS3 reduced virus growth. Thus, our study demonstrated that cellular components comprising of MVB and exocytic pathways proteins are involved in BTV replication in ovine cells.

  • Viruses, Vol. 7, Pages 2358-2377: Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s) Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

  • Human coronavirus OC43 (HCoV-OC43) is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses.

  • Viruses, Vol. 7, Pages 2334-2357: Mechanisms of Cancer Cell Killing by the Adenovirus E4orf4 Protein

  • During adenovirus (Ad) replication the Ad E4orf4 protein regulates progression from the early to the late phase of infection. However, when E4orf4 is expressed alone outside the context of the virus it induces a non-canonical mode of programmed cell death, which feeds into known cell death pathways such as apoptosis or necrosis, depending on the cell line tested. E4orf4-induced cell death has many interesting and unique features including a higher susceptibility of cancer cells to E4orf4-induced cell killing compared with normal cells, caspase-independence, a high degree of evolutionary conservation of the signaling pathways, a link to perturbations of the cell cycle, and involvement of two distinct cell death programs, in the nucleus and in the cytoplasm. Several E4orf4-interacting proteins including its major partners, protein phosphatase 2A (PP2A) and Src family kinases, contribute to induction of cell death. The various features of E4orf4-induced cell killing as well as studies to decipher the underlying mechanisms are described here. Many explanations for the cancer specificity of E4orf4-induced cell death have been proposed, but a full understanding of the reasons for the different susceptibility of cancer and normal cells to killing by E4orf4 will require a more detailed analysis of the complex E4orf4 signaling network. An improved understanding of the mechanisms involved in this unique mode of programmed cell death may aid in design of novel E4orf4-based cancer therapeutics.

  • Viruses, Vol. 7, Pages 2321-2333: Alphaviruses in Gene Therapy

  • Alphavirus vectors present an attractive approach for gene therapy applications due to the rapid and simple recombinant virus particle production and their broad range of mammalian host cell transduction. Mainly three types of alphavirus vectors, namely naked RNA, recombinant particles and DNA/RNA layered vectors, have been subjected to preclinical studies with the goal of achieving prophylactic or therapeutic efficacy, particularly in oncology. In this context, immunization with alphavirus vectors has provided protection against challenges with tumor cells. Moreover, alphavirus intratumoral and systemic delivery has demonstrated substantial tumor regression and significant prolonged survival rates in various animal tumor models. Recent discoveries of the strong association of RNA interference and disease have accelerated gene therapy based approaches, where alphavirus-based gene delivery can play an important role.

  • Viruses, Vol. 7, Pages 2308-2320: Increased Viral Dissemination in the Brain and Lethality in MCMV-Infected, Dicer-Deficient Neonates

  • Among Herpesviruses, Human Cytomegalovirus (HCMV or HHV-5) represents a major threat during congenital or neonatal infections, which may lead to encephalitis with serious neurological consequences. However, as opposed to other less prevalent pathogens, the mechanisms and genetic susceptibility factors for CMV encephalitis are poorly understood. This lack of information considerably reduces the prognostic and/or therapeutic possibilities. To easily monitor the effects of genetic defects on brain dissemination following CMV infection we used a recently developed in vivo mouse model based on the neonatal inoculation of a MCMV genetically engineered to express Luciferase. Here, we further validate this protocol for live imaging, and demonstrate increased lethality associated with viral infection and encephalitis in mutant mice lacking Dicer activity. Our data indicate that miRNAs are important players in the control of MCMV pathogenesis and suggest that miRNA-based endothelial functions and integrity are crucial for CMV encephalitis.

  • Viruses, Vol. 7, Pages 2288-2307: Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems

  • To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as“good” and “bad” BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA waslower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields.

  • Viruses, Vol. 7, Pages 2268-2287: Celecoxib Inhibits the Lytic Activation of Kaposi’s Sarcoma-Associated Herpesvirus through Down-Regulation of RTA Expression by Inhibiting the Activation of p38 MAPK

  • Kaposi’s sarcoma associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). KSHV’s lytic replication cycle is critical for the pathogenesis of KSHV-associated diseases. Despite recent progress in thedevelopment of treatments for KSHV associated malignancies, these therapies are not completely efficacious and cause side effects. Therefore, more effective therapies with antiviral agents against KSHV are urgently needed. In this study, we identified celecoxib as an antiviral agent against KSHV. Our data suggest that celecoxib inhibits the lytic activation of KSHV through the down-regulation of the expression of the lytic switch protein, replication and transcription activator (RTA), by inhibiting the activation of p38 MAPK. Therefore, celecoxib may provide a candidate inhibitor for the therapeutic research of KSHV-related malignancies.

  • Viruses, Vol. 7, Pages 2230-2267: Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

  • Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

  • Viruses, Vol. 7, Pages 2210-2229: NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

  • Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  • Viruses, Vol. 7, Pages 2185-2209: Genetic Characterization of the Tick-Borne Orbiviruses

  • The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.

  • Viruses, Vol. 7, Pages 2168-2184: A Phylogeographic Investigation of African Monkeypox

  • Monkeypox is a zoonotic disease caused by a virus member of the genus Orthopoxvirus and is endemic to Central and Western African countries. Previous work has identified two geographically disjuct clades of monkeypox virus based on the analysis of a few genomes coupled with epidemiological and clinical analyses; however, environmental and geographic causes of this differentiation have not been explored. Here, we expand previous phylogenetic studies by analyzing a larger set of monkeypox virus genomes originating throughout Sub-Saharan Africa to identify possible biogeographic barriers associated with genetic differentiation; and projected ecological niche models onto environmental conditions at three periods in the past to explore the potential role of climate oscillations in the evolution of the two primary clades. Analyses supported the separation of the Congo Basin and West Africa clades; the Congo Basin clade shows much shorter branches, which likely indicate a more recent diversification of isolates within this clade. The area between the Sanaga and Cross Rivers divides the two clades and the Dahomey Gap seems to have also served as a barrier within the West African clade. Contraction of areas with suitable environments for monkeypox virus during the Last Glacial Maximum, suggests that the Congo Basin clade of monkeypox virus experienced a severe bottleneck and has since expanded its geographic range.

  • Viruses, Vol. 7, Pages 2147-2167: BST2/Tetherin Inhibition of Alphavirus Exit

  • Alphaviruses such as chikungunya virus (CHIKV) and Semliki Forest virus (SFV) are small enveloped RNA viruses that bud from the plasma membrane. Tetherin/BST2 is an interferon-induced host membrane protein that inhibits the release of many enveloped viruses via direct tethering of budded particles to the cell surface. Alphaviruses have highly organized structures and exclude host membrane proteins from the site of budding, suggesting that their release might be insensitive to tetherin inhibition. Here, we demonstrated that exogenously-expressed tetherin efficiently inhibited the release of SFV and CHIKV particles from host cells without affecting virus entry and infection. Alphavirus release was also inhibited by the endogenous levels of tetherin in HeLa cells. While rubella virus (RuV) and dengue virus (DENV) have structural similarities to alphaviruses, tetherin inhibited the release of RuV but not DENV. We found that two recently identified tetherin isoforms differing in length at the N-terminus exhibited distinct capabilities in restricting alphavirus release. SFV exit was efficiently inhibited by the long isoform but not the short isoform of tetherin, while both isoforms inhibited vesicular stomatitis virus exit. Thus, in spite of the organized structure of the virus particle, tetherin specifically blocks alphavirus release and shows an interesting isoform requirement.

  • Viruses, Vol. 7, Pages 2126-2146: Genome Variability and Gene Content in Chordopoxviruses: Dependence on Microsatellites

  • To investigate gene loss in poxviruses belonging to the Chordopoxvirinae subfamily, we assessed the gene content of representative members of the subfamily, and determined whether individual genes present in each genome were intact, truncated, or fragmented. When nonintact genes were identified, the early stop mutations (ESMs) leading to gene truncation or fragmentation were analyzed. Of all the ESMs present in these poxvirus genomes, over 65% co-localized with microsatellites—simple sequence nucleotide repeats. On average, microsatellites comprise 24% of the nucleotide sequence of these poxvirus genomes. These simple repeats have been shown to exhibit high rates of variation, and represent a target for poxvirus protein variation, gene truncation, and reductive evolution.

  • Viruses, Vol. 7, Pages 2099-2125: Baculovirus-mediated Gene Delivery and RNAi Applications

  • Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  • Viruses, Vol. 7, Pages 2074-2098: Can Plant Viruses Cross the Kingdom Border and Be Pathogenic to Humans?

  • Phytoviruses are highly prevalent in plants worldwide, including vegetables and fruits. Humans, and more generally animals, are exposed daily to these viruses, among which several are extremely stable. It is currently accepted that a strict separation exists between plant and vertebrate viruses regarding their host range and pathogenicity, and plant viruses are believed to infect only plants. Accordingly, plant viruses are not considered to present potential pathogenicity to humans and other vertebrates. Notwithstanding these beliefs, there are many examples where phytoviruses circulate and propagate in insect vectors. Several issues are raised here that question if plant viruses might further cross the kingdom barrier to cause diseases in humans. Indeed, there is close relatedness between some plant and animal viruses, and almost identical gene repertoires. Moreover, plant viruses can be detected in non-human mammals and humans samples, and there are evidence of immune responses to plant viruses in invertebrates, non-human vertebrates and humans, and of the entry of plant viruses or their genomes into non-human mammal cells and bodies after experimental exposure. Overall, the question raised here is unresolved, and several data prompt the additional extensive study of the interactions between phytoviruses and non-human mammals and humans, and the potential of these viruses to cause diseases in humans.

  • Viruses, Vol. 7, Pages 2057-2073: Enhancement of Herpes Simplex Virus (HSV) Infection by Seminal Plasma and Semen Amyloids Implicates a New Target for the Prevention of HSV Infection

  • Human herpesviruses cause different infectious diseases, resulting in world-wide health problems. Sexual transmission is a major route for the spread of both herpes simplex virus-1 (HSV-1) and -2. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa and, thereby, initiates viral replication. Previously, we demonstrated that the amyloid fibrils semenogelin (SEM) and semen-derived enhancer of viral infection (SEVI), and seminal plasma (SP) augment cytomegalovirus infection (Tang et al., J. Virol 2013). Whether SEM or SEVI amyloids or SP could also enhance other herpesvirus infections has not been examined. In this study, we found that the two amyloids as well as SP strongly enhance both HSV-1 and -2 infections in cell culture. Along with SP, SEM and SEVI amyloids enhanced viral entry and increased infection rates by more than 10-fold, as assessed by flow cytometry assay and fluorescence microscopy. Viral replication was increased by about 50- to 100-fold. Moreover, viral growth curve assays showed that SEM and SEVI amyloids, as well as SP, sped up the kinetics of HSV replication such that the virus reached its replicative peak more quickly. The interactions of SEM, SEVI, and SP with HSVs are direct. Furthermore, we discovered that the enhancing effects of SP, SEM, and SEVI can be significantly reduced by heparin, a sulfated polysaccharide with an anionic charge. It is probable that heparin abrogates said enhancing effects by interfering with the interaction of the viral particle and the amyloids, which interaction results in the binding of the viral particles and both SEM and SEVI.

  • Viruses, Vol. 7, Pages 2030-2056: Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

  • There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important fornon-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.

  • Viruses, Vol. 7, Pages 2014-2029: Frequent Infection of Human Cancer Xenografts with Murine Endogenous Retroviruses in Vivo

  • Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infectedwith XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effectson xenograft growth potential with significant consequences for interpretation of many xenograft studies.

  • Viruses, Vol. 7, Pages 2006-2013: Serological Evidence of Hantavirus Infection in Apparently Healthy People from Rural and Slum Communities in Southern Chile

  • Hantavirus disease in America has been recognizable because of its rapid progression in clinical cases, occurrence in previously healthy young adults, and high case fatality rate. Hantavirus disease has been proposed now to define the diversity of clinical manifestations. Since 1995, a total of 902 cases of hantavirus pulmonary syndrome have been reported in Chile, caused by Andes virus (ANDV), with overall fatality of 32%. This report describes the sero-epidemiology of hantavirus in apparently healthy people in rural and urban slum communities from southern Chile. Ten of 934 samples yielded a positive result resulting in a seroprevalence of 1.07% (95% confidence intervals: 0.05%–2.0%). A higher proportion of positive samples was found among individuals from rural villages (1.3%) and slums (1.5%) compared with farms (0.5%). Seropositivity was associated with age (p = 0.011), low education level (p = 0.006) and occupations linked to the household (homemaker, retired, or student) (p = 0.016). No evidence of infection was found in 38 sigmodontinae rodents trapped in the peri-domestic environment. Our findings highlight that exposure risk was associated with less documented risk factors, such as women in slum and rural villages, and the occurrence of infection that may have presented as flu-like illness that did not require medical attention or was misdiagnosed.

  • Viruses, Vol. 7, Pages 1987-2005: A Piscine Birnavirus Induces Inhibition of Protein Synthesis in CHSE-214 Cells Primarily through the Induction of eIF2α Phosphorylation

  • Inhibition of protein synthesis represents one of the antiviral mechanisms employed by cells and it is also used by viruses for their own propagation. To what extent members of the Birnaviridae family employ such strategies is not well understood. Here we use a type-strain of the Aquabirnavirus, infectious pancreatic necrosis virus (IPNV), to investigate this phenomenon in vitro. CHSE-214 cells were infected with IPNV and at 3, 12, 24, and 48 hours post infection (hpi) before the cells were harvested and labeled with S35 methionine to assess protein synthesis. eIF2α phosphorylation was examined by Western blot while RT-qPCR was used to assess virus replication and the expression levels of IFN-α, Mx1 and PKR. Cellular responses to IPNV infection were assessed by DNA laddering, Caspase-3 assays and flow cytometry. The results show that the onset and kinetics of eIF2α phosphorylation was similar to that of protein synthesis inhibition as shown by metabolic labeling. Increased virus replication and virus protein formation was observed by 12 hpi, peaking at 24 hpi. Apoptosis was induced in a small fraction (1−2%) of IPNV-infected CHSE cells from 24 hpi while necrotic/late apoptotic cells increased from 10% by 24 hpi to 59% at 48 hpi, as shown by flow cytometry. These results were in accordance with a small decline in cell viability by 24hpi, dropping below 50% by 48 hpi. IPNV induced IFN-α mRNA upregulation by 24 hpi while no change was observed in the expression of Mx1 and PKR mRNA. Collectively, these findings show that IPNV induces inhibition of protein synthesis in CHSE cells through phosphorylation of eIF2α with minimal involvement of apoptosis. The anticipation is that protein inhibition is used by the virus to evade the host innate antiviral responses.

  • Viruses, Vol. 7, Pages 1975-1986: Evaluating Environmental Persistence and Disinfection of the Ebola Virus Makona Variant

  • Background: The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. Methods: EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. Results: EBOV/Mak persisted longer on steel and plastic surfaces (192 hours) than cotton (aamp;amp;lt;24 hours). Dilute sodium hypochlorite (0.01% and 0.1%) showed little antiviral action, whereas 0.5% and 1% sodium hypochlorite solutions demonstrated recoverable virus at one minute but sterilized surfaces in five minutes. Disinfection with 67% ethanol did not fully clear infectious virions from 3/9 carriers at 1 minute but sterilized all carriers at 5 and 10 minutes. Conclusions: Sodium hypochlorite and ethanol effectively decontaminate EBOV/Mak suspended in a simulated organic load; however, selection of concentration and contact time proves critical.

  • Viruses, Vol. 7, Pages 1960-1974: Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

  • Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host’s immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

  • Viruses, Vol. 7, Pages 1927-1959: Insect-Specific Flaviviruses: A Systematic Review of Their Discovery, Host Range, Mode of Transmission, Superinfection Exclusion Potential and Genomic Organization

  • There has been a dramatic increase in the number of insect-specific flaviviruses (ISFs) discovered in the last decade. Historically, these viruses have generated limited interest due to their inability to infect vertebrate cells. This viewpoint has changed in recent years because some ISFs have been shown to enhance or suppress the replication of medically important flaviviruses in co-infected mosquito cells. Additionally, comparative studies between ISFs and medically important flaviviruses can provide a unique perspective as to why some flaviviruses possess the ability to infect and cause devastating disease in humans while others do not. ISFs have been isolated exclusively from mosquitoes in nature but the detection of ISF-like sequences in sandflies and chironomids indicates that they may also infect other dipterans. ISFs can be divided into two distinct phylogenetic groups. The first group currently consists of approximately 12 viruses and includes cell fusing agent virus, Kamiti River virus and Culex flavivirus. These viruses are phylogenetically distinct from all other known flaviviruses. The second group, which is apparently not monophyletic, currently consists of nine viruses and includes Chaoyang virus, Nounané virus and Lammi virus. These viruses phylogenetically affiliate with mosquito/vertebrate flaviviruses despite their apparent insect-restricted phenotype. This article provides a review of the discovery, host range, mode of transmission, superinfection exclusion ability and genomic organization of ISFs. This article also attempts to clarify the ISF nomenclature because some of these viruses have been assigned more than one name due to their simultaneous discoveries by independent research groups.

  • Viruses, Vol. 7, Pages 1902-1926: Archaeal Viruses Multiply: Temporal Screening in a Solar Saltern

  • Hypersaline environments around the world are dominated by archaea and their viruses. To date, very little is known about these viruses and their interaction with the host strains when compared to bacterial and eukaryotic viruses. We performed the first culture-dependent temporal screening of haloarchaeal viruses and their hosts in the saltern of Samut Sakhon, Thailand, during two subsequent years (2009, 2010). Altogether we obtained 36 haloarchaeal virus isolates and 36 archaeal strains, significantly increasing the number of known archaeal virus isolates. Interestingly, the morphological distribution of our temporal isolates (head-tailed, pleomorphic, and icosahedral membrane-containing viruses) was similar to the outcome of our previous spatial survey supporting the observations of a global resemblance of halophilic microorganisms and their viruses. Myoviruses represented the most abundant virus morphotype with strikingly broad host ranges. The other viral morphotypes (siphoviruses, as well as pleomorphic and icosahedral internal membrane-containing viruses) were more host-specific. We also identified a group of Halorubrum strains highly susceptible to numerous different viruses (up to 26). This high virus sensitivity, the abundance of broad host range viruses, and the maintenance of infectivity over a period of one year suggest constant interplay of halophilic microorganisms and their viruses within an extreme environment.

  • Viruses, Vol. 7, Pages 1871-1901: The Role of Merkel Cell Polyomavirus and Other Human Polyomaviruses in Emerging Hallmarks of Cancer

  • Polyomaviruses are non-enveloped, dsDNA viruses that are common in mammals, including humans. All polyomaviruses encode the large T-antigen and small t-antigen proteins that share conserved functional domains, comprising binding motifs for the tumor suppressors pRb and p53, and for protein phosphatase 2A, respectively. At present, 13 different human polyomaviruses are known, and for some of them their large T-antigen and small t-antigen have been shown to possess oncogenic properties in cell culture and animal models, while similar functions are assumed for the large T- and small t-antigen of other human polyomaviruses. However, so far the Merkel cell polyomavirus seems to be the only human polyomavirus associated with cancer. The large T- and small t-antigen exert their tumorigenic effects through classical hallmarks of cancer: inhibiting tumor suppressors, activating tumor promoters, preventing apoptosis, inducing angiogenesis and stimulating metastasis. This review elaborates on the putative roles of human polyomaviruses in some of the emerging hallmarks of cancer. The reciprocal interactions between human polyomaviruses and the immune system response are discussed, a plausible role of polyomavirus-encoded and polyomavirus-induced microRNA in cancer is described, and the effect of polyomaviruses on energy homeostasis and exosomes is explored. Therapeutic strategies against these emerging hallmarks of cancer are also suggested.

  • Viruses, Vol. 7, Pages 1853-1870: A Current Overview of the Papaya meleira virus, an Unusual Plant Virus

  • Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease, which is characterized by a spontaneous exudation of fluid and aqueous latex from the papaya fruit and leaves. The latex oxidizes after atmospheric exposure, resulting in a sticky feature on the fruit from which the name of the disease originates. PMeV is an isometric virus particle with a double-stranded RNA (dsRNA) genome of approximately 12 Kb. Unusual for a plant virus, PMeV particles are localized on and linked to the polymers present in the latex. The ability of the PMeV to inhabit such a hostile environment demonstrates an intriguing interaction of the virus with the papaya. A hypersensitivity response is triggered against PMeV infection, and there is a reduction in the proteolytic activity of papaya latex during sticky disease. In papaya leaf tissues, stress responsive proteins, mostly calreticulin and proteasome-related proteins, are up regulated and proteins related to metabolism are down-regulated. Additionally, PMeV modifies the transcription of several miRNAs involved in the modulation of genes related to the ubiquitin-proteasome system. Until now, no PMeV resistant papaya genotype has been identified and roguing is the only viral control strategy available. However, a single inoculation of papaya plants with PMeV dsRNA delayed the progress of viral infection.

  • Viruses, Vol. 7, Pages 1832-1852: In Ovo Delivery of CpG DNA Reduces Avian Infectious Laryngotracheitis Virus Induced Mortality and Morbidity

  • Endosomal toll-like receptor-21 and -9 sense CpG DNA activating production of pro-inflammatory mediators with antimicrobial effects. Here, we investigated the induction of antiviral response of in ovo delivered CpG DNA against infectious laryngotracheitis virus (ILTV) infection. We found that in ovo delivered CpG DNA significantly reduces ILTV infection pre-hatch correlating with the expression of IL-1β and increase of macrophages in lungs. As assessed in vitro, CpG DNA stimulated avian macrophages could be a potential source of IL-1β and other pro-inflammatory mediators. Since we also found that in ovo CpG DNA delivery maintains increased macrophages in the lungs post-hatch, we infected the chickens on the day of hatch with ILTV. We found that in ovo delivered CpG DNA significantly reduces mortality and morbidity resulting from ILTV infection encountered post-hatch. Thus, CpG DNA can be a candidate innate immune stimulant worthy of further investigation for the control of ILTV infection in chickens.

  • Viruses, Vol. 7, Pages 1823-1831: Analysis of HDAC6 and BAG3-Aggresome Pathways in African Swine Fever Viral Factory Formation

  • African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes.

  • Viruses, Vol. 7, Pages 1804-1822: A Suggested New Bacteriophage Genus, “Kp34likevirus”, within the Autographivirinae Subfamily of Podoviridae

  • Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called“Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.

  • Viruses, Vol. 7, Pages 1726-1803: The Evolution of Poxvirus Vaccines

  • After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

  • Viruses, Vol. 7, Pages 1700-1725: Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

  • The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

  • Viruses, Vol. 7, Pages 1685-1699: Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry)

  • Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.

  • Viruses, Vol. 7, Pages 1668-1684: Human Pirh2 is A Novel Inhibitor of Prototype Foamy Virus Replication

  • Prototype foamy virus (PFV) is a member of the unconventional and nonpathogenic retroviruses. PFV causes lifelong chronic infections, which are partially attributable to a number of host cell factors that restrict viral replication. Herein, we identified human p53-induced RING-H2 protein (Pirh2) as a novel inhibitor of prototype foamy virus. Overexpression of Pirh2 decreased the replication of PFV, whereas knockdown of Pirh2 with specific siRNA increased PFV replication. Dual-luciferase assays and coimmunoprecipitation demonstrated that Pirh2 negatively influences the Tas-dependent transcriptional activation of the PFV long terminal repeat (LTR) and internal promoter (IP) by interacting with the transactivator Tas and down-regulating its expression. In addition, the viral inhibitory function of Pirh2 is N-terminal and RING domain dependent. Together, these results indicated that Pirh2 suppresses PFV replication by negatively impacting its transactivator Tas and the transcription of two viral promoters, which may contribute to the latency of PFV infection.

  • Viruses, Vol. 7, Pages 1667: Addendum: Kaźmierczak, Z.; Górski, A.; Dąbrowska, K. Facing Antibiotic Resistance: Staphylococcus aureus Phages as a Medical Tool. Viruses 2014, 6, 2551-2570.

  • The authors would like to add the following Acknowledgements section: “Acknowledgements This work was funded by the National Science Centre in Poland grant UMO-2012/05/E/NZ6/03314.” [...]

  • Viruses, Vol. 7, Pages 1651-1666: Tenth International Foamy Virus Conference 2014–Achievements and Perspectives

  • For the past two decades, scientists from around the world, working on different aspects of foamy virus (FV) research, have gathered in different research institutions almost every two years to present their recent results in formal talks, to discuss their ongoing studies informally, and to initiate fruitful collaborations. In this report we review the 2014 anniversary conference to share the meeting summary with the virology community and hope to arouse interest by other researchers to join this exciting field. The topics covered included epidemiology, virus molecular biology, and immunology of FV infection in non-human primates, cattle, and humans with zoonotic FV infections, as well as recent findings on endogenous FVs. Several topics focused on virus replication and interactions between viral and cellular proteins. Use of FV in biomedical research was highlighted with presentations on using FV vectors for gene therapy and FV proteins as scaffold for vaccine antigen presentation. On behalf of the FV community, this report also includes a short tribute to commemorate Prof. Axel Rethwilm, one of the leading experts in the field of retrovirology and foamy viruses, who passed away 29 July 2014.

  • Viruses, Vol. 7, Pages 1642-1650: Presence of Poly(A) Tails at the 3'-Termini of Some mRNAs of a Double-Stranded RNA Virus, Southern Rice Black-Streaked Dwarf Virus

  • Southern rice black-streaked dwarf virus (SRBSDV), a new member of the genus Fijivirus, is a double-stranded RNA virus known to lack poly(A) tails. We now showed that some of SRBSDV mRNAs were indeed polyadenylated at the 3' terminus in plant hosts, and investigated the nature of 3' poly(A) tails. The non-abundant presence of SRBSDV mRNAs bearing polyadenylate tails suggested that these viral RNA were subjected to polyadenylation-stimulated degradation. The discovery of poly(A) tails in different families of viruses implies potentially a wide occurrence of the polyadenylation-assisted RNA degradation in viruses.

  • Viruses, Vol. 7, Pages 1627-1641: Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

  • Kaposi’s sarcoma-associated herpesvirus (KSHV) is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS). KSHV G protein-coupled receptor (vGPCR) is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at theplasma membrane is crucial for vGPCR mediated signaling.

  • Viruses, Vol. 7, Pages 1613-1626: Apigenin Restricts FMDV Infection and Inhibits Viral IRES Driven Translational Activity

  • Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild ruminants that is caused by FMD virus (FMDV). FMD outbreaks have occurred in livestock-containing regions worldwide. Apigenin, which is a flavonoid naturally existing in plant, possesses various pharmacological effects, including anti-inflammatory, anticancer, antioxidant and antiviral activities. Results show that apigenin can inhibit FMDV-mediated cytopathogenic effect and FMDV replication in vitro. Further studies demonstrate the following: (i) apigenin inhibits FMDV infection at the viral post-entry stage; (ii) apigenin does not exhibit direct extracellular virucidal activity; and (iii) apigenin interferes with the translational activity of FMDV driven by internal ribosome entry site. Studies on applying apigein in vivo are required for drug development and further identification of potential drug targets against FDMV infection.

  • Viruses, Vol. 7, Pages 1599-1612: Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus

  • Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterialβ-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture aswell as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

  • Viruses, Vol. 7, Pages 1578-1598: Eradication of HIV-1 from the Macrophage Reservoir: An Uncertain Goal?

  • Human immunodeficiency virus type 1 (HIV-1) establishes latency in resting memory CD4+ T cells and cells of myeloid lineage. In contrast to the T cells, cells of myeloid lineage are resistant to the HIV-1 induced cytopathic effect. Cells of myeloid lineage including macrophages are present in anatomical sanctuaries making them a difficult drug target. In addition, the long life span of macrophages as compared to the CD4+ T cells make them important viral reservoirs in infected individuals especially in the late stage of viral infection where CD4+ T cells are largely depleted. In the past decade, HIV-1 persistence in resting CD4+ T cells has gained considerable attention. It is currently believed that rebound viremia following cessation of combination anti-retroviral therapy (cART) originates from this source. However, the clinical relevance of this reservoir has been questioned. It is suggested that the resting CD4+ T cells are only one source of residual viremia and other viral reservoirs such as tissue macrophages should be seriously considered. In the present review we will discuss how macrophages contribute to the development of long-lived latent reservoirs and how macrophages can be used as a therapeutic target in eradicating latent reservoir.

  • Viruses, Vol. 7, Pages 1558-1577: Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease

  • Enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease with especially severe neurologic complications, which mainly account for fatalities from this disease. To date, the pathogenesis of EV71 in the central neurons system has remained unclear. Cytokine-mediated immunopathogenesis and nervous tissue damage by virus proliferation are two widely speculated causes of the neurological disease. To further study the pathogenesis, we identified a common epitope (co-epitope) between EV71 VP1 and human mediator complex subunit 25 (MED25) highly expressed in brain stem. A monoclonal antibody (2H2) against the co-epitope was prepared, and its interaction with MED25 was examined by ELISA, immunofluorescence assay and Western blot in vitro and by live small animal imaging in vivo. Additionally, 2H2 could bind to both VP1 and MED25 with the affinity constant (Kd) of 10−7 M as determined by the ForteBio Octet System. Intravenously injected 2H2 was distributed in brain stem of mice after seven days of EV71 infection. Interestingly, 2H2-like antibodies were detected in the serum of EV71-infected patients. These findings suggest that EV71 infection induces the production of antibodies that can bind to autoantigens expressed in nervous tissue and maybe further trigger autoimmune reactions resulting in neurological disease.

  • Viruses, Vol. 7, Pages 1540-1557: Viral Interference with Functions of the Cellular Receptor Tyrosine Phosphatase CD45

  • The receptor tyrosine phosphatase CD45 is expressed on the surface of almost all cells of hematopoietic origin. CD45 functions are central to the development of T cells and determine the threshold at which T and B lymphocytes can become activated. Given this pivotal role of CD45 in the immune system, it is probably not surprising that viruses interfere with the activity of CD45 in lymphocytes to dampen the immune response and that they also utilize this molecule to accomplish their replication cycle. Here we report what is known about the interaction of viral proteins with CD45. Moreover, we debate putative interactions of viruses with CD45 in myeloid cells and the resulting consequences—subjects that remain to be investigated. Finally, we summarize the evidence that pathogens were the driving force for the evolution of CD45.
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