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

Table of Contents for this page:

  • Current Issue
  • Current Issue of Viruses


  • Viruses, Vol. 8, Pages 293: Ubiquitin in Influenza Virus Entry and Innate Immunity

  • Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle.

  • Viruses, Vol. 8, Pages 296: Griffithsin: An Antiviral Lectin with Outstanding Therapeutic Potential

  • Griffithsin (GRFT), an algae-derived lectin, is one of the most potent viral entry inhibitors discovered to date. It is currently being developed as a microbicide with broad-spectrum activity against several enveloped viruses. GRFT can inhibit human immunodeficiency virus (HIV) infection at picomolar concentrations, surpassing the ability of most anti-HIV agents. The potential to inhibit other viruses as well as parasites has also been demonstrated. Griffithsin’s antiviral activity stems from its ability to bind terminal mannoses present in high-mannose oligosaccharides and crosslink these glycans on the surface of the viral envelope glycoproteins. Here, we review structural and biochemical studies that established mode of action and facilitated construction of GRFT analogs, mechanisms that may lead to resistance, and in vitro and pre-clinical results that support the therapeutic potential of this lectin.

  • Viruses, Vol. 8, Pages 295: The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus

  • Human cytomegalovirus is a ubiquitousβ-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a verysimilar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane.

  • Viruses, Vol. 8, Pages 291: Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model

  • African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV). There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4) virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi) showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN)-γ responses, or specific cytokine profiles) and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms.

  • Viruses, Vol. 8, Pages 294: Measles to the Rescue: A Review of Oncolytic Measles Virus

  • Oncolytic virotherapeutic agents are likely to become serious contenders in cancer treatment. The vaccine strain of measles virus is an agent with an impressive range of oncolytic activity in pre-clinical trials with increasing evidence of safety and efficacy in early clinical trials. This paramyxovirus vaccine has a proven safety record and is amenable to careful genetic modification in the laboratory. Overexpression of the measles virus (MV) receptor CD46 in many tumour cells may direct the virus to preferentially enter transformed cells and there is increasing awareness of the importance of nectin-4 and signaling lymphocytic activation molecule (SLAM) in oncolysis. Successful attempts to retarget MV by inserting genes for tumour-specific ligands to antigens such as carcinoembryonic antigen (CEA), CD20, CD38, and by engineering the virus to express synthetic microRNA targeting sequences, and“blinding” the virus to the natural viral receptors are exciting measures to increase viral specificity and enhance the oncolytic effect. Sodium iodine symporter (NIS) can also be expressed by MV, which enables in vivo tracking of MV infection. Radiovirotherapy using MV-NIS, chemo-virotherapy to convert prodrugs to their toxic metabolites, and immune-virotherapy including incorporating antibodies against immune checkpoint inhibitors can also increase the oncolytic potential. Anti-viral host immune responses are a recognized barrier to the success of MV, and approaches such as transportingMV to the tumour sites by carrier cells, are showing promise. MV Clinical trials are producing encouraging preliminary results in ovarian cancer, myeloma and cutaneous non-Hodgkin lymphoma, and the outcome of currently open trials in glioblastoma multiforme, mesothelioma and squamous cell carcinomaare eagerly anticipated.

  • Viruses, Vol. 8, Pages 290: The Role of Nuclear Antiviral Factors against Invading DNA Viruses: The Immediate Fate of Incoming Viral Genomes

  • In recent years, it has been suggested that host cells exert intrinsic mechanisms to control nuclear replicating DNA viruses. This cellular response involves nuclear antiviral factors targeting incoming viral genomes. Herpes simplex virus-1 (HSV-1) is the best-studied model in this context, and it was shown that upon nuclear entry HSV-1 genomes are immediately targeted by components of promyelocytic leukemia nuclear bodies (PML-NBs) and the nuclear DNA sensor IFI16 (interferon gamma inducible protein 16). Based on HSV-1 studies, together with limited examples in other viral systems, these phenomena are widely believed to be a common cellular response to incoming viral genomes, although formal evidence for each virus is lacking. Indeed, recent studies suggest that the case may be different for adenovirus infection. Here we summarize the existing experimental evidence for the roles of nuclear antiviral factors against incoming viral genomes to better understand cellular responses on a virus-by-virus basis. We emphasize that cells seem to respond differently to different incoming viral genomes and discuss possible arguments for and against a unifying cellular mechanism targeting the incoming genomes of different virus families.

  • Viruses, Vol. 8, Pages 292: In Vitro and In Vivo Models for the Study of Human Polyomavirus Infection

  • Developments of genome amplification techniques have rapidly expanded the family of human polyomaviruses (PyV). Following infection early in life, PyV persist in their hosts and are generally of no clinical consequence. High-level replication of PyV can occur in patients under immunosuppressive or immunomodulatory therapy and causes severe clinical entities, such as progressive multifocal leukoencephalopathy, polyomavirus-associated nephropathy or Merkel cell carcinoma. The characterization of known and newly-discovered human PyV, their relationship to human health, and the mechanisms underlying pathogenesis remain to be elucidated. Here, we summarize the most widely-used in vitro and in vivo models to study the PyV-host interaction, pathogenesis and anti-viral drug screening. We discuss the strengths and limitations of the different models and the lessons learned.

  • Viruses, Vol. 8, Pages 289: Cutthroat Trout Virus—Towards a Virus Model to Support Hepatitis E Research

  • Cutthroat trout virus (CTV) is a non-pathogenic fish virus belonging to the Hepeviridae family, and it is distantly related to hepatitis E virus (HEV). Here, we report the development of an efficient cell culture system where CTV can consistently replicate to titers never observed before with a hepevirus. By using the rainbow trout gill (RTGill-W1) cell line, CTV reaches 1010 geq/mL intracellularly and 109 geq/mL extracellularly within 5–6 days in culture. We additionally established a qPCR system to investigate CTV infectivity, and developed a specific antibody directed against the viral capsid protein encoded by ORF2. With these methods, we were able to follow the progressive accumulation of viral RNA and the capsid protein, and their intracellular distribution during virus replication. Virus progeny purified through iodixanol density gradients indicated—that similar to HEV—CTV produced in cell culture is also lipid-associated. The lack of an efficient cell culture system has greatly impeded studies with HEV, a majorhuman pathogen that causes hepatitis worldwide. Although several cell culture systems have recently been established, the replication efficiency of HEV is not robust enough to allow studies on different aspects of the virus replication cycle. Therefore, a surrogate virus that can replicate easily and efficiently in cultured cells would be helpful to boost research studies with hepeviruses. Due to its similarities, but also its key differences to HEV, CTV represents a promising tool to elucidate aspects of the replication cycle of Hepeviridae in general, and HEV in particular.

  • Viruses, Vol. 8, Pages 281: Hepatitis B Virus Protein X Induces Degradation of Talin-1

  • In the infected human hepatocyte, expression of the hepatitis B virus (HBV) accessory protein X (HBx) is essential to maintain viral replication in vivo. HBx critically interacts with the host damaged DNA binding protein 1 (DDB1) and the associated ubiquitin ligase machinery, suggesting that HBx functions by inducing the degradation of host proteins. To identify such host proteins, we systematically analyzed the HBx interactome. One HBx interacting protein, talin-1 (TLN1), was proteasomally degraded upon HBx expression. Further analysis showed that TLN1 levels indeed modulate HBV transcriptional activity in an HBx-dependent manner. This indicates that HBx-mediated TLN1 degradation is essential and sufficient to stimulate HBV replication. Our data show that TLN1 can act as a viral restriction factor that suppresses HBV replication, and suggest that the HBx relieves this restriction by inducing TLN1 degradation.

  • Viruses, Vol. 8, Pages 288: Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

  • Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

  • Viruses, Vol. 8, Pages 287: 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

  • West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of thehost mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  • Viruses, Vol. 8, Pages 286: Maporal Hantavirus Causes Mild Pathology in Deer Mice (Peromyscus maniculatus)

  • Rodent-borne hantaviruses can cause two human diseases with many pathological similarities: hantavirus cardiopulmonary syndrome (HCPS) in the western hemisphere and hemorrhagic fever with renal syndrome in the eastern hemisphere. Each virus is hosted by specific reservoir species without conspicuous disease. HCPS-causing hantaviruses require animal biosafety level-4 (ABSL-4) containment, which substantially limits experimental research of interactions between the viruses and their reservoir hosts. Maporal virus (MAPV) is a South American hantavirus not known to cause disease in humans, thus it can be manipulated under ABSL-3 conditions. The aim of this study was to develop an ABSL-3 hantavirus infection model using the deer mouse (Peromyscus maniculatus), the natural reservoir host of Sin Nombre virus (SNV), and a virus that is pathogenic in another animal model to examine immune response of a reservoir host species. Deer mice were inoculated with MAPV, and viral RNA was detected in several organs of all deer mice during the 56 day experiment. Infected animals generated both nucleocapsid-specific and neutralizing antibodies. Histopathological lesions were minimal to mild with the peak of the lesions detected at 7–14 days postinfection, mainly in the lungs, heart, and liver. Low to modest levels of cytokine gene expression were detected in spleens and lungs of infected deer mice, and deer mouse primary pulmonary cells generated with endothelial cell growth factors were susceptible to MAPV with viral RNA accumulating in the cellular fraction compared to infected Vero cells. Most features resembled that of SNV infection of deer mice, suggesting this model may be an ABSL-3 surrogate for studying the host response of a New World hantavirus reservoir.

  • Viruses, Vol. 8, Pages 284: Gene Regulation and Quality Control in Murine Polyomavirus Infection

  • Murine polyomavirus (MPyV) infects mouse cells and is highly oncogenic in immunocompromised hosts and in other rodents. Its genome is a small, circular DNA molecule of just over 5000 base pairs and it encodes only seven polypeptides. While seemingly simply organized, this virus has adopted an unusual genome structure and some unusual uses of cellular quality control pathways that, together, allow an amazingly complex and varied pattern of gene regulation. In this review we discuss how MPyV leverages these various pathways to control its life cycle.

  • Viruses, Vol. 8, Pages 285: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

  • BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletionthat is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involvingthe asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail.

  • Viruses, Vol. 8, Pages 283: Mutagenic Effects of Ribavirin on Hepatitis E Virus—Viral Extinction versus Selection of Fitness-Enhancing Mutations

  • Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis.

  • Viruses, Vol. 8, Pages 282: The Immune Response in Measles: Virus Control, Clearance and Protective Immunity

  • Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10–14 days. The first appearance of the disease is a 2–3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4+ and CD8+ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity.

  • Viruses, Vol. 8, Pages 275: The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

  • The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells.

  • Viruses, Vol. 8, Pages 276: Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

  • Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.

  • Viruses, Vol. 8, Pages 274: Morbillivirus Experimental Animal Models: Measles Virus Pathogenesis Insights from Canine Distemper Virus

  • Morbilliviruses share considerable structural and functional similarities. Even though disease severity varies among the respective host species, the underlying pathogenesis and the clinical signs are comparable. Thus, insights gained with one morbillivirus often apply to the other members of the genus. Since the Canine distemper virus (CDV) causes severe and often lethal disease in dogs and ferrets, it is an attractive model to characterize morbillivirus pathogenesis mechanisms and to evaluate the efficacy of new prophylactic and therapeutic approaches. This review compares the cellular tropism, pathogenesis, mechanisms of persistence and immunosuppression of the Measles virus (MeV) and CDV. It then summarizes the contributions made by studies on the CDV in dogs and ferrets to our understanding of MeV pathogenesis and to vaccine and drugs development.

  • Viruses, Vol. 8, Pages 273: A Single Lineage of Hepatitis E Virus Causes Both  Outbreaks and Sporadic Hepatitis in Sudan

  • Few studies have reported sporadic hepatitis E virus (HEV) infections during non‐outbreak periods in Africa. In this study, the prevalence of HEV infection in Sudan was investigated in 432 patients with acute hepatitis from 12 localities in North Kordofan, and from 152 patients involved in smaller outbreaks of hepatitis in the neighbouring Darfur. HEV infection was diagnosed in 147 (25%) patients: 98 from Kordofan and 49 from Darfur. The mortality was 10%; six of the patients who died from the infection were pregnant women. HEV RNA was detected by quantitative real‐time polymerase chain reaction (RT‐qPCR) in 38 (26%) patients: 22 from Kordofan and 16 from Darfur.Partial open reading frame (ORF) 1 and ORF2 were sequenced from HEV from nine and three patients, respectively. Phylogenetic analysis showed that the Sudanese strains belonged to genotype 1 (HEV1), and confirmed the segregation of African HEV1 strains into one branch divergent from Asian HEV1. It also revealed that the Sudanese strains from this study and from an outbreak in 2004 formed a separate clade with a common ancestor, distinct from strains from the neighbouring Chad and Egypt. This HEV strain has thus spread in a large area of Sudan, where it has caused both sporadic hepatitis E and outbreaks from at least 2004 and onwards. These data demonstrate that hepatitis E is a constant, on‐going public health problem in Sudan and that there is a need for hepatitis E surveillance, outbreak preparedness, and general improvements of the sanitation in these remote areas of the country.

  • Viruses, Vol. 8, Pages 272: A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection

  • Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long‐distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA‐dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3.

  • Viruses, Vol. 8, Pages 271: Atypical Porcine Pestivirus: A Possible Cause of Congenital Tremor Type A‐II in Newborn Piglets

  • Congenital tremor type A‐II in piglets has been regarded as a transmissible disease since the 1970s, possibly caused by a very recently‐described virus: atypical porcine pestivirus (APPV). Here, we describe several strains of APPV in piglets with clinical signs of congenital tremor (10 of 10 farms tested). Piglets ona farm with no history of congenital tremor were PCR‐negative for the virus. To demonstrate a causal relationship between APPV and disease, three gilts were inoculated via intramuscular injection at day 32 of pregnancy. In two of the three litters, vertical transmission of the virus occurred. Clinical signs of congenital tremor were observed in APPV‐infected newborns, yet also two asymptomatic carriers were among the offspring. Piglets of one litter were PCR‐negative for the virus, and these piglets were all without congenital tremors. Long‐term follow up of farm piglets born with congenital tremors showed that the initially high viremia in serum declines at five months of age, but shedding of the virus in feces continues, which explains why the virus remains present at affected farms and causes new outbreaks. We conclude that trans‐placental transmission of APPV and subsequent infection of the fetuses is a very likely cause of congenital tremor type A‐II in piglets.

  • Viruses, Vol. 8, Pages 270: Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes

  • During the past ten years, several new hepatitis E viruses (HEVs) have been identified in various animal species. In parallel, the number of reports of autochthonous hepatitis E in Western countries has increased as well, raising the question of what role these possible animal reservoirs play in human infections. The aim of this review is to present the recent discoveries of animal HEVs and their classification within the Hepeviridae family, their zoonotic and species barrier crossing potential, and possible use as models to study hepatitis E pathogenesis. Lastly, this review describes the transmission pathways identified from animal sources.

  • Viruses, Vol. 8, Pages 269: Multi-Omics Studies towards Novel Modulators of Influenza A Virus–Host Interaction

  • Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV–host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV–host interactions, which might be more effective than the currently available anti-influenza therapeutics.

  • Viruses, Vol. 8, Pages 267: Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

  • Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  • Viruses, Vol. 8, Pages 264: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

  • The major threat for cassava cultivation on the Indian subcontinent is cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses which are bipartite begomoviruses with DNA A and DNA B components. Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) cause CMD in India. Two isolates of SLCMV infected the cassava cultivar Sengutchi in the fields near Malappuram and Thiruvananthapuram cities of Kerala State, India. The Malappuram isolate was persistent when maintained in the Madurai Kamaraj University (MKU, Madurai, Tamil Nadu, India) greenhouse, whereas the Thiruvananthapuram isolate did not persist. The recovered cassava plants with the non-persistent SLCMV, which were maintained vegetative in quarantine in the University of Basel (Basel, Switzerland) greenhouse, displayed re-emergence of CMD after a six-month period. Interestingly, these plants did not carry SLCMV but carried ICMV. It is interpreted that the field-collected, SLCMV-infected cassava plants were co-infected with low levels of ICMV. The loss of SLCMV in recovered cassava plants, under greenhouse conditions, then facilitated the re-emergence of ICMV. The partial dimer clones of the persistent and non-persistent isolates of SLCMV and the re-emerged isolate of ICMV were infective in Nicotiana benthamiana upon agroinoculation. Studies on pseudo-recombination between SLCMV and ICMV in N. benthamiana provided evidence for trans-replication of ICMV DNA B by SLCMV DNA A.

  • Viruses, Vol. 8, Pages 265: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

  • Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells.

  • Viruses, Vol. 8, Pages 260: Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

  • Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques,and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development.

  • Viruses, Vol. 8, Pages 268: Molecular Characterization of a Trisegmented Mycovirus from the Plant Pathogenic Fungus Colletotrichum gloeosporioides

  • A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1–3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5′- and 3′-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1.

  • Viruses, Vol. 8, Pages 266: Vesicular Nucleo-Cytoplasmic Transport—Herpesviruses as Pioneers in Cell Biology

  • Herpesviruses use a vesicle-mediated transfer of intranuclearly assembled nucleocapsids through the nuclear envelope (NE) for final maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex (NEC), conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane (INM) by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane (ONM). Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the NE has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport.

  • Viruses, Vol. 8, Pages 263: Mechanistic Study of Common Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutations with K103N and Y181C Substitutions

  • Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for human immunodeficiency type 1 virus (HIV-1) infections. However, their effectiveness can be hampered by the emergence of resistant mutations. To aid in designing effective NNRTIs against the resistant mutants, it is important to understand the resistance mechanism of the mutations. Here, we investigate the mechanism of the two most prevalent NNRTI-associated mutations with K103N or Y181C substitution. Virus and reverse transcriptase (RT) with K103N/Y188F, K103A, or K103E substitutions and with Y181F, Y188F, or Y181F/Y188F substitutions were employed to study the resistance mechanism of the K103N and Y181C mutants, respectively. Results showed that the virus and RT with K103N/Y188F substitutions displayed similar resistance levels to the virus and RT with K103N substitution versus NNRTIs. Virus and RT containing Y181F, Y188F, or Y181F/Y188F substitution exhibited either enhanced or similar susceptibility to NNRTIs compared with the wild type (WT) virus. These results suggest that the hydrogen bond between N103 and Y188 may not play an important role in the resistance of the K103N variant to NNRTIs. Furthermore, the results from the studies with the Y181 or Y188 variant provide the direct evidence that aromaticπ–π stacking plays a crucial role in the binding of NNRTIs to RT.

  • Viruses, Vol. 8, Pages 258: Capsid-Targeted Viral Inactivation: A Novel Tactic for Inhibiting Replication in Viral Infections

  • Capsid-targeted viral inactivation (CTVI), a conceptually powerful new antiviral strategy, is attracting increasing attention from researchers. Specifically, this strategy is based on fusion between the capsid protein of a virus and a crucial effector molecule, such as a nuclease (e.g., staphylococcal nuclease, Barrase, RNase HI), lipase, protease, or single-chain antibody (scAb). In general, capsid proteins have a major role in viral integration and assembly, and the effector molecule used in CTVI functions to degrade viral DNA/RNA or interfere with proper folding of viral key proteins, thereby affecting the infectivity of progeny viruses. Interestingly, such a capsid–enzyme fusion protein is incorporated into virions during packaging. CTVI is more efficient compared to other antiviral methods, and this approach is promising for antiviral prophylaxis and therapy. This review summarizes the mechanism and utility of CTVI and provides some successful applications of this strategy, with the ultimate goal of widely implementing CTVI in antiviral research.

  • Viruses, Vol. 8, Pages 262: Zebra Alphaherpesviruses (EHV-1 and EHV-9): Genetic Diversity, Latency and Co-Infections

  • Alphaherpesviruses are highly prevalent in equine populations and co-infections with more than one of these viruses’ strains frequently diagnosed. Lytic replication and latency with subsequent reactivation, along with new episodes of disease, can be influenced by genetic diversity generated by spontaneous mutation and recombination. Latency enhances virus survival by providing an epidemiological strategy for long-term maintenance of divergent strains in animal populations. The alphaherpesviruses equine herpesvirus 1 (EHV-1) and 9 (EHV-9) have recently been shown to cross species barriers, including a recombinant EHV-1 observed in fatal infections of a polar bear and Asian rhinoceros. Little is known about the latency and genetic diversity of EHV-1 and EHV-9, especially among zoo and wild equids. Here, we report evidence of limited genetic diversity in EHV-9 in zebras, whereas there is substantial genetic variability in EHV-1. We demonstrate that zebras can be lytically and latently infected with both viruses concurrently. Such a co-occurrence of infection in zebras suggests that even relatively slow-evolving viruses such as equine herpesviruses have the potential to diversify rapidly by recombination. This has potential consequences for the diagnosis of these viruses and their management in wild and captive equid populations.

  • Viruses, Vol. 8, Pages 253: Transmission of Hepatitis E Virus in Developing Countries

  • Hepatitis E virus (HEV), an RNA virus of the Hepeviridae family, has marked heterogeneity. While all five HEV genotypes can cause human infections, genotypes HEV-1 and -2 infect humans alone, genotypes HEV-3 and -4 primarily infect pigs, boars and deer, and genotype HEV-7 primarily infects dromedaries. The global distribution of HEV has distinct epidemiological patterns based on ecology and socioeconomic factors. In resource-poor countries, disease presents as large-scale waterborne epidemics, and few epidemics have spread through person-to-person contact; however, endemic diseases within these countries can potentially spread through person-to-person contact or fecally contaminated water and foods. Vertical transmission of HEV from infected mother to fetus causes high fetal and perinatal mortality. Other means of transmission, such as zoonotic transmission, can fluctuate depending upon the region and strain of the virus. For instance, zoonotic transmission can sometimes play an insignificant role in human infections, such as in India, where human and pig HEV infections are unrelated. However, recently China and Southeast Asia have experienced a zoonotic spread of HEV-4 from pigs to humans and this has become the dominant mode of transmission of hepatitis E in eastern China. Zoonotic HEV infections in humans occur by eating undercooked pig flesh, raw liver, and sausages; through vocational contact; or via pig slurry, which leads to environmental contamination of agricultural products and seafood. Lastly, blood transfusion-associated HEV infections occur in many countries and screening of donors for HEV RNA is currently under serious consideration. To summarize, HEV genotypes 1 and 2 cause epidemic and endemic diseases in resource poor countries, primarily spreading through contaminated drinking water. HEV genotypes 3 and 4 on the other hand, cause autochthonous infections in developed, and many developing countries, by means of a unique zoonotic food-borne transmission.

  • Viruses, Vol. 8, Pages 261: Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection

  • Chronic hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are major causes of liver disease and hepatocellular carcinoma worldwide. Despite the presence of an efficient preventive vaccine, more than 250 million patients are chronically infected with HBV. Current antivirals effectively control but only rarely cure chronic infection. While the molecular biology of the two viruses has been characterized in great detail, the absence of robust cell culture models for HBV and/or HDV infection has limited the investigation of virus-host interactions. Native hepatoma cell lines do not allow viral infection, and the culture of primary hepatocytes, the natural host cell for the viruses, implies a series of constraints restricting the possibilities of analyzing virus-host interactions. Recently, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key HBV/HDV cell entry factor has opened the door to a new era of investigation, as NTCP-overexpressing hepatoma cells acquire susceptibility to HBV and HDV infections. In this review, we summarize the major cell culture models for HBV and HDV infection, discuss their advantages and limitations and highlight perspectives for future developments.

  • Viruses, Vol. 8, Pages 257: Orchestrating the Selection and Packaging of Genomic RNA by Retroviruses: An Ensemble of Viral and Host Factors

  • Infectious retrovirus particles contain two copies of unspliced viral RNA that serve as the viral genome. Unspliced retroviral RNA is transcribed in the nucleus by the host RNA polymerase II and has three potential fates: (1) it can be spliced into subgenomic messenger RNAs (mRNAs) for the translation of viral proteins; or it can remain unspliced to serve as either (2) the mRNA for the translation of Gag and Gag–Pol; or (3) the genomic RNA (gRNA) that is packaged into virions. The Gag structural protein recognizes and binds the unspliced viral RNA to select it as a genome, which is selected in preference to spliced viral RNAs and cellular RNAs. In this review, we summarize the current state of understanding about how retroviral packaging is orchestrated within the cell and explore potential new mechanisms based on recent discoveries in the field. We discuss the cis-acting elements in the unspliced viral RNA and the properties of the Gag protein that are required for their interaction. In addition,we discuss the role of host factors in influencing the fate of the newly transcribed viral RNA, current models for how retroviruses distinguish unspliced viral mRNA from viral genomic RNA, and the possible subcellular sites of genomic RNA dimerization and selection by Gag. Although this review centers primarily on the wealth of data available for the alpharetrovirus Rous sarcoma virus, in which a discrete RNA packaging sequence has been identified, we have also summarized the cis- and trans-acting factors as well as the mechanisms governing gRNA packaging of other retroviruses for comparison.

  • Viruses, Vol. 8, Pages 250: The Host Cell Receptors for Measles Virus and Their Interaction with the Viral Hemagglutinin (H) Protein

  • The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles“blind” to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces.

  • Viruses, Vol. 8, Pages 259: Markers for Ongoing or Previous Hepatitis E Virus Infection Are as Common in Wild Ungulates as in Humans in Sweden

  • Hepatitis E virus (HEV) is a human pathogen with zoonotic spread, infecting both domestic and wild animals. About 17% of the Swedish population is immune to HEV, but few cases are reported annually, indicating that most infections are subclinical. However, clinical hepatitis E may also be overlooked. For identified cases, the source of infection is mostly unknown. In order to identify whether HEV may be spread from wild game, the prevalence of markers for past and/or ongoing infection was investigated in sera and stool samples collected from 260 hunted Swedish wild ungulates. HEV markers were found in 43 (17%) of the animals. The most commonly infected animal was moose (Alces alces) with 19 out of 69 animals (28%) showing HEV markers, followed by wild boar (Sus scrofa) with 21 out of 139 animals (15%), roe deer (Capreolus capreolus) with 2 out of 30 animals, red deer (Cervus elaphus) with 1 out of 15 animals, and fallow deer (Dama dama) 0 out of 7 animals. Partial open reading frame 1 (ORF1) of the viral genomes from the animals were sequenced and compared with those from 14 endemic human cases. Phylogenetic analysis revealed that three humans were infected with HEV strains similar to those from wild boar. These results indicate that wild animals may be a source of transmission to humans and could be an unrecognized public health concern.

  • Viruses, Vol. 8, Pages 256: Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

  • Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The“psi” (Ψ) element within the 5′-untranslated region (5′UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity.

  • Viruses, Vol. 8, Pages 255: Arms Race between Enveloped Viruses and the Host ERAD Machinery

  • Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed to potentiate or inhibit viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells.

  • Viruses, Vol. 8, Pages 254: Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

  • Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

  • Viruses, Vol. 8, Pages 247: Binding of RNA by the Nucleoproteins of Influenza Viruses A and B

  • This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

  • Viruses, Vol. 8, Pages 246: On the Selective Packaging of Genomic RNA by HIV-1

  • Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

  • Viruses, Vol. 8, Pages 251: Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex

  • The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pavethe path towards the structure-based design and optimization of next-generation RdRp inhibitors.

  • Viruses, Vol. 8, Pages 248: The Life-Cycle of the HIV-1 Gag–RNA Complex

  • Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles.

  • Viruses, Vol. 8, Pages 249: Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

  • The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

  • Viruses, Vol. 8, Pages 252: Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line

  • Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.

  • Viruses, Vol. 8, Pages 245: Analysis of the Prevalence of HTLV-1 Proviral DNA in Cervical Smears and Carcinomas from HIV Positive and Negative Kenyan Women

  • The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV−ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck®) and cytology. This showed 22/113 (19.5%) of LBC’s from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV−ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07–26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV−ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p aamp;amp;lt; 0.05) and smoking (p aamp;amp;lt; 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear.

  • Viruses, Vol. 8, Pages 244: A Decrease in Temperature and Humidity Precedes Human Rhinovirus Infections in a Cold Climate

  • Both temperature and humidity may independently or jointly contribute to the risk of human rhinovirus (HRV) infections, either through altered survival and spread of viruses in the environment or due to changes in host susceptibility. This study examined the relationship between short-term variations in temperature and humidity and the risk of HRV infections in a subarctic climate. We conducted a case-crossover study among conscripts (n = 892) seeking medical attention due to respiratory symptoms during their military training and identified 147 HRV cases by real-time PCR. An average temperature, a decline in daily ambient temperature and absolute humidity (AH) during the three preceding days of the onset (hazard period) and two reference periods (a week prior and after the onset) were obtained. The average daily temperature preceding HRV infections was−9.9 ± 4.9 °C and the average AH was 2.2 ± 0.9 g/m3. An average (odds ratios (OR) 1.07 (95% confidence interval (CI) 1.00–1.15)) and maximal (OR 1.08 (1.01–1.17)) change in temperature increased the risk of HRV infections by 8% per 1 °C decrease. An average (OR 1.20 (CI 1.03–1.40)) and maximal decrease (OR 1.13 (CI 0.96–1.34)) in AH increased the risk of HRV infection by 13% and 20% per 0.5 g/m3 decrease. A higher average temperature during the three preceding days was positively associated with HRV infections (OR 1.07 (CI 1.00–1.15)). A decrease rather than low temperature and humidity per se during the preceding few days increases the risk of HRV infections in a cold climate. The information is applicable to populations residing in cold climates for appropriate personal protection and prevention of adverse health effects.

  • Viruses, Vol. 8, Pages 241: Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface

  • The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.

  • Viruses, Vol. 8, Pages 243: Foamy Virus Protein—Nucleic Acid Interactions during Particle Morphogenesis

  • Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches.

  • Viruses, Vol. 8, Pages 242: How Polyomaviruses Exploit the ERAD Machinery to Cause Infection

  • To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases.

  • Viruses, Vol. 8, Pages 240: Evaluation of the Cross-Protective Efficacy of a Chimeric Porcine Reproductive and Respiratory Syndrome Virus Constructed Based on Two Field Strains

  • One of the major hurdles to porcine reproductive and respiratory syndrome (PRRS) vaccinology is the limited or no cross-protection conferred by current vaccines. To overcome this challenge, a PRRS chimeric virus (CV) was constructed using an FL12-based cDNA infectious clone in which open reading frames (ORFs) 3–4 and ORFs 5–6 were replaced with the two Korean field isolates K08-1054 and K07-2273,respectively. This virus was evaluated as a vaccine candidate to provide simultaneous protection against two genetically distinct PRRS virus (PRRSV) strains. Thirty PRRS-negative three-week-old pigs were divided into five groups and vaccinated with CV, K08-1054, K07-2273, VR-2332, or a mock inoculum. At 25 days post-vaccination (dpv), the pigs in each group were divided further into two groups and challenged with either K08-1054 or K07-2273. All of the pigs were observed until 42 dpv and were euthanizedfor pathological evaluation. Overall, the CV-vaccinated group exhibited higher levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-12 (IL-12) expression and of serum virus-neutralizing antibodies compared with the other groups after vaccination and also demonstrated better protection levels against both viruses compared with the challenge control group. Based on these results, it was concluded that CV might be an effective vaccine model that can confer a broader range of cross-protection to various PRRSV strains.

  • Viruses, Vol. 8, Pages 232: Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays

  • Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes.

  • Viruses, Vol. 8, Pages 237: A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

  • Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

  • Viruses, Vol. 8, Pages 239: From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

  • In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.

  • Viruses, Vol. 8, Pages 238: Influenza NA and PB1 Gene Segments Interact during the Formation of Viral Progeny: Localization of the Binding Region within the PB1 Gene

  • The influenza A virus genome comprises eight negative-sense viral RNAs (vRNAs) that form individual ribonucleoprotein (RNP) complexes. In order to incorporate a complete set of each of these vRNAs, the virus uses a selective packaging mechanism that facilitates co-packaging of specific gene segments but whose molecular basis is still not fully understood. Recently, we used a competitive transfection model where plasmids encoding the A/Puerto Rico/8/34 (PR8) and A/Udorn/307/72 (Udorn) PB1 gene segments were competed to show that the Udorn PB1 gene segment is preferentially co-packaged into progeny virions with the Udorn NA gene segment. Here we created chimeric PB1 genes combining both Udorn and PR8 PB1 sequences to further define the location within the Udorn PB1 gene that drives co-segregation of these genes and show that nucleotides 1776–2070 of the PB1 gene are crucial for preferential selection. In vitro assays examining specific interactions between Udorn NA vRNA and purified vRNAs transcribed from chimeric PB1 genes also supported the importance of this region in the PB1-NA interaction. Hence, this work identifies an association between viral genes that are co-selected during packaging. It also reveals a region potentially important in the RNP-RNP interactions within the supramolecular complex that is predicted to form prior to budding to allow one of each segment to be packaged in the viral progeny. Our study lays thefoundation to understand the co-selection of specific genes, which may be critical to the emergence of new viruses with pandemic potential.

  • Viruses, Vol. 8, Pages 236: Assessment of Domestic Pigs, Wild Boars and Feral Hybrid Pigs as Reservoirs of Hepatitis E Virus in Corsica, France

  • In Corsica, extensive pig breeding systems allow frequent interactions between wild boars and domestic pigs, which are suspected to act as reservoirs of several zoonotic diseases including hepatitis E virus (HEV). In this context, 370 sera and 166 liver samples were collected from phenotypically characterized as pure or hybrid wild boars, between 2009 and 2012. In addition, serum and liver from 208 domestic pigs belonging to 30 farms were collected at the abattoir during the end of 2013. Anti-HEV antibodies were detected in 26% (21%–31.6%) of the pure wild boar, 43.5% (31%–56.7%) of hybrid wild boar and 88% (82.6%–91.9%) of the domestic pig sera. In addition, HEV RNA was detected in five wild boars, three hybrid wild boars and two domestic pig livers tested. Our findings provide evidence that both domestic pig and wild boar (pure and hybrid) act as reservoirs of HEV in Corsica, representing an important zoonotic risk for Corsican hunters and farmers but also for the large population of consumers of raw pig liver specialties produced in Corsica. In addition, hybrid wild boars seem to play an important ecological role in the dissemination of HEV between domestic pig and wild boar populations, unnoticed to date, that deserves further investigation.

  • Viruses, Vol. 8, Pages 234: Performance Evaluation of the VIDAS® Measles IgG Assay and Its Diagnostic Value for Measuring IgG Antibody Avidity in Measles Virus Infection

  • The objective of this study is primarily to compare the performance of the VIDAS® Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost® Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA® (Microimmune). The sensitivity and the agreement of the VIDAS® Measles IgG assay compared to the Enzygnost® Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA® assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS® Measles IgG assayto measure anti-measles virus IgG antibody avidity with the help of the VIDAS® CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI) aamp;amp;lt; 0.3 and a strong avidity as an AI aamp;amp;gt; 0.6. The VIDAS® Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects.

  • Viruses, Vol. 8, Pages 235: The Host RNAs in Retroviral Particles

  • As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive.

  • Viruses, Vol. 8, Pages 233: Quantification of HEV RNA by Droplet Digital PCR

  • The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p aamp;amp;lt; 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types.

  • Viruses, Vol. 8, Pages 231: Subversion of the Immune Response by Rabies Virus

  • Rabies has affected mankind for several centuries and is one of the oldest known zoonoses. It is peculiar how little is known regarding the means by which rabies virus (RABV) evades the immune response and kills its host. This review investigates the complex interplay between RABV and the immune system, including the various means by which RABV evades, or advantageously utilizes, the host immune response in order to ensure successful replication and spread to another host. Different factors that influence immune responses—including age, sex, cerebral lateralization and temperature—are discussed, with specific reference to RABV and the effects on host morbidity and mortality. We also investigate the role of apoptosis and discuss whether it is a detrimental or beneficial mechanism of the host’s response to infection. The various RABV proteins and their roles in immune evasion are examined in depth with reference to important domains and the downstream effects of these interactions. Lastly, an overview of the means by which RABV evades important immune responses is provided. The research discussed in this review will be important in determining the roles of the immune response during RABV infections as well as to highlight important therapeutic target regions and potential strategies for rabies treatment.

  • Viruses, Vol. 8, Pages 230: Inoculation of Goats, Sheep, and Horses with MERS-CoV Does Not Result in Productive Viral Shedding

  • The Middle East respiratory syndrome coronavirus (MERS-CoV) was first recognized in 2012 and can cause severe disease in infected humans. Dromedary camels are the reservoir for the virus, although, other than nasal discharge, these animals do not display any overt clinical disease. Data from in vitro experiments suggest that other livestock such as sheep, goats, and horses might also contribute to viral transmission, although field data has not identified any seropositive animals. In order to understand if these animals could be infected, we challenged young goats and horses and adult sheep with MERS-CoV by intranasal inoculation. Minimal or no virus shedding was detected in all of the animals. During the four weeks following inoculation, neutralizing antibodies were detected in the young goats, but not in sheep or horses.

  • Viruses, Vol. 8, Pages 227: A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

  • Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology.

  • Viruses, Vol. 8, Pages 229: Role of Envelopment in the HEV Life Cycle

  • Hepatitis E virus (HEV), an enterically transmitted hepatotropic virus, was thought to be non-enveloped for decades. However, recent studies have revealed that the virus circulating in the patient’s blood is completely cloaked in host membranes and resistant to neutralizing antibodies. The discovery of this novel enveloped form of HEV has raised a series of questions about the fundamental biology of HEV and the way this virus, which has been understudied in the past, interacts with its host. Here, we review recent advances towards understanding this phenomenon and discuss its potential impact on various aspects of the HEV life cycle and immunity.

  • Viruses, Vol. 8, Pages 219: Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

  • The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

  • Viruses, Vol. 8, Pages 228: Ins and Outs of Multipartite Positive-Strand RNA Plant Viruses: Packaging versus Systemic Spread

  • Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the“life aspects” of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant.

  • Viruses, Vol. 8, Pages 226: Addressing the Challenges of Hepatitis C Virus Resistance and Treatment Failure

  • Chronic hepatitis C is a major cause of chronic liver disease, including liver cirrhosis and hepatocellular carcinoma. The development of direct-acting antivirals (DAAs) revolutionized hepatitis C virus (HCV) treatment by offering genuine prospects for the first comprehensive cure of a chronic viral infection in humans. While antiviral resistance is a significant limitation for interferon-based therapies, resistance and treatment failure still appear to be present in a small fraction of patients even in state-of-the-art DAA combination therapies. Therefore, treatment failure and resistance still remain a clinical challenge for the management of patients not responding to DAAs. In this special issue of Viruses on HCV drug resistance, mechanisms of antiviral resistance for different classes of antiviral drugs are described. Furthermore, the detection and monitoring of resistance in clinical practice, the clinical impact of resistance in different patient groups and strategies to prevent and address resistance and treatment failure using complementary antiviral strategies are reviewed.

  • Viruses, Vol. 8, Pages 222: Treatment of HEV Infection in Patients with a Solid-Organ Transplant and Chronic Hepatitis

  • Hepatitis E virus (HEV) infection can cause hepatic and extra-hepatic manifestations. Treatment of HEV infection has been thoroughly studied in solid-organ-transplant patients who have developed a chronic HEV infection. In this review, we report on our current knowledge regarding treatment of HEV infection.

  • Viruses, Vol. 8, Pages 220: Spinal Cord Ventral Horns and Lymphoid Organ Involvement in Powassan Virus Infection in a Mouse Model

  • Powassan virus (POWV) belongs to the family Flaviviridae and is a member of the tick-borne encephalitis serogroup. Transmission of POWV from infected ticks to humans has been documented in the USA, Canada, and Russia, causing fatal encephalitis in 10% of human cases and significant neurological sequelae in survivors. We used C57BL/6 mice to investigate POWV infection and pathogenesis. After footpad inoculation, infected animals exhibited rapid disease progression and 100% mortality. Immunohistochemistry and immunofluorescence revealed a very strong neuronal tropism of POWV infection. The central nervous system infection appeared as a meningoencephalitis with perivascular mononuclear infiltration and microglial activation in the brain, and a poliomyelitis-like syndrome with high level of POWV antigen at the ventral horn of the spinal cord. Pathological studies also revealed substantial infection of splenic macrophages by POWV, which suggests that the spleen plays a more important role in pathogenesis than previously realized. This report provides a detailed description of the neuroanatomical distribution of the lesions produced by POWV infection in C57BL/6 mice.

  • Viruses, Vol. 8, Pages 223: What Do We Know about How Hantaviruses Interact with Their Different Hosts?

  • Hantaviruses, like other members of the Bunyaviridae family, are emerging viruses that are able to cause hemorrhagic fevers. Occasional transmission to humans is due to inhalation of contaminated aerosolized excreta from infected rodents. Hantaviruses are asymptomatic in their rodent or insectivore natural hosts with which they have co-evolved for millions of years. In contrast, hantaviruses cause different pathologies in humans with varying mortality rates, depending on the hantavirus species and its geographic origin. Cases of hemorrhagic fever with renal syndrome (HFRS) have been reported in Europe and Asia, while hantavirus cardiopulmonary syndromes (HCPS) are observed in the Americas. In some cases, diseases caused by Old World hantaviruses exhibit HCPS-like symptoms. Although the etiologic agents of HFRS were identified in the early 1980s, the way hantaviruses interact with their different hosts still remains elusive. What are the entry receptors? How do hantaviruses propagate in the organism and how do they cope with the immune system? This review summarizes recent data documenting interactions established by pathogenic and nonpathogenic hantaviruses with their natural or human hosts that could highlight their different outcomes.

  • Viruses, Vol. 8, Pages 225: Tomato Infection by Whitefly-Transmitted Circulative and Non-Circulative Viruses Induce Contrasting Changes in Plant Volatiles and Vector Behaviour

  • Virus infection frequently modifies plant phenotypes, leading to changes in behaviour and performance of their insect vectors in a way that transmission is enhanced, although this may not always be the case. Here, we investigated Bemisia tabaci response to tomato plants infected by Tomato chlorosis virus (ToCV), a non-circulative-transmitted crinivirus, and Tomato severe rugose virus (ToSRV), a circulative-transmitted begomovirus. Moreover, we examined the role of visual and olfactory cues in host plant selection by both viruliferous and non-viruliferous B. tabaci. Visual cues alone were assessed as targets for whitefly landing by placing leaves underneath a Plexiglas plate. A dual-choice arena was used to assess whitefly response to virus-infected and mock-inoculated tomato leaves under light and dark conditions. Thereafter, we tested the whitefly response to volatiles using an active air-flow Y-tube olfactometer, and chemically characterized the blends using gas chromatography coupled to mass spectrometry. Visual stimuli tests showed that whiteflies, irrespective of their infectious status, always preferred to land on virus-infected rather than on mock-inoculated leaves. Furthermore, whiteflies had no preference for either virus-infected or mock-inoculated leaves under dark conditions, but preferred virus-infected leaves in the presence of light. ToSRV-infection promoted a sharp decline in the concentration of some tomato volatiles, while an increase in the emission of some terpenes after ToCV infection was found. ToSRV-viruliferous whiteflies preferred volatiles emitted from mock-inoculated plants, a conducive behaviour to enhance virus spread, while volatiles from ToCV-infected plants were avoided by non-viruliferous whiteflies, a behaviour that is likely detrimental to the secondary spread of the virus. In conclusion, the circulative persistent begomovirus, ToSRV, seems to have evolved together with its vector B. tabaci to optimise its own spread. However, this type of virus-induced manipulation of vector behaviour was not observed for the semi persistent crinivirus, ToCV, which is not specifically transmitted by B. tabaci and has a much less intimate virus-vector relationship.

  • Viruses, Vol. 8, Pages 221: Telomere Length, Proviral Load and Neurologic Impairment in HTLV-1 and HTLV-2-Infected Subjects

  • Short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that analysis of telomere length (TL) in human T-cell lymphotropic virus (HTLV) infection and HTLV-associated neuropathy might provide clues to the etiology of HTLV-associated disease and viral dynamics. A subset of 45 human T-cell lymphotropic virus type 1 (HTLV-1), 45 human T-cell lymphotropic virus type 2 (HTLV-2), and 45 seronegative subjects was selected from the larger HTLV Outcomes Study (HOST) cohort, matched on age, sex and race/ethnicity. Telomere-to-single-copy gene (T/S) ratio (a measure of TL) and HTLV-1 and HTLV-2 proviral loads were measured in peripheral blood mononuclear cells (PBMCs) using quantitative PCR (qPCR). Vibration sensation measured by tuning fork during neurologic examinations performed as part of the HOST study allowed for an assessment of peripheral neuropathy. TL was compared between groups using t-tests, linear and logisticregression. Mean T/S ratio was 1.02 ± 0.16 in HTLV-1, 1.03 ± 0.17 in HTLV-2 and 0.99 ± 0.18 in HTLV seronegative subjects (p = 0.322). TL was not associated with HTLV-1 or -2 proviral load. Shorter TL was significantly associated with impaired vibration sense in the HTLV-2 positive group only. Overall, we found no evidence that telomere length was affected by chronic HTLV-1 and HTLV-2 infection. That TL was only associated with peripheral neuropathy in the HTLV-2-positive group is intriguing, but should be interpreted cautiously. Studies with larger sample size and telomere length measurement in lymphocyte subsets may clarify the relationship between TL and HTLV-infection.

  • Viruses, Vol. 8, Pages 224: Homage to Richard M. Elliott

  • In the last 25 years, the scientific and public attention paid to bunyaviruses has increased considerably.[...]

  • Viruses, Vol. 8, Pages 218: Experimental Approaches to Study Genome Packaging of Influenza A Viruses

  • The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level.

  • Viruses, Vol. 8, Pages 211: Hepatitis E Seroprevalence in Europe: A Meta-Analysis

  • There have been large numbers of studies on anti-HEV IgG seroprevalence in Europe, however, the results of these studies have produced high variability of seroprevalence rates, making interpretation increasingly problematic. Therefore, the aim of this study was to develop a clearer understanding of anti-HEV IgG seroprevalence in Europe and identify risk groups for HEV exposure by a meta-analysis of published studies. Methods: All European HEV-seroprevalence studies from 2003 to 2015 were reviewed. Data were stratified by assay, geographical location, and patient cohort (general population, patients with HIV, solid-organ transplant recipients, chronic liver disease patients, and individuals in contact with swine/wild animals). Data were pooled using a mixed-effects model. Results: Four hundred thirty-two studies were initially identified, of which 73 studies were included in the analysis. Seroprevalence estimates ranged from 0.6% to 52.5%, increased with age, but were unrelated to gender. General population seroprevalence varied depending on assays: Wantai (WT): 17%, Mikrogen (MG): 10%, MP-diagnostics (MP): 7%, DiaPro: 4%, Abbott 2%. The WT assay reported significantly higher seroprevalence rates across all cohorts (p aamp;amp;lt; 0.001). Individuals in contact with swine/wild animals had significantly higher seroprevalence rates than the general population, irrespective of assay (p aamp;amp;lt; 0.0001). There was no difference between any other cohorts. The highest seroprevalence was observed in France (WT: 32%, MP: 16%) the lowest in Italy (WT: 7.5%, MP 0.9%). Seroprevalence varied between and within countries. The observed heterogeneity was attributed to geographical region (23%), assay employed (23%)and study cohort (7%). Conclusion: Seroprevalcence rates primarily depend on the seroassy that is used, followed by the geographical region and study cohort. Seroprevalence is higher in individuals exposed to swine and/or wild animals, and increases with age.

  • Viruses, Vol. 8, Pages 212: Hepatitis E Pathogenesis

  • Although most hepatitis E virus (HEV) infections are asymptomatic, some can be severe, causing fulminant hepatitis and extra-hepatic manifestations, including neurological and kidney injuries. Chronic HEV infections may also occur in immunocompromised patients. This review describes how our understanding of the pathogenesis of HEV infection has progressed in recent years.

  • Viruses, Vol. 8, Pages 214: Fluorescent and Bioluminescent Reporter Myxoviruses

  • The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo.

  • Viruses, Vol. 8, Pages 217: Erratum: Smither, S.; et al. Effectiveness of Four Disinfectants against Ebola Virus on Different Materials. Viruses 2016, 8, 185

  • The Viruses Editorial Office wishes to notify its readers of corrections in [1].[...]

  • Viruses, Vol. 8, Pages 215: How Active Are Porcine Endogenous Retroviruses (PERVs)?

  • Porcine endogenous retroviruses (PERVs) represent a risk factor if porcine cells, tissues, or organs were to be transplanted into human recipients to alleviate the shortage of human transplants; a procedure called xenotransplantation. In contrast to human endogenous retroviruses (HERVs), which are mostly defective and not replication-competent, PERVs are released from normal pig cells and are infectious. PERV-A and PERV-B are polytropic viruses infecting cells of several species, among them humans; whereas PERV-C is an ecotropic virus infecting only pig cells. Virus infection was shown in co-culture experiments, but also in vivo, in the pig, leading to de novo integration of proviruses in certain organs. This was shown by measurement of the copy number per cell, finding different numbers in different organs. In addition, recombinations between PERV-A and PERV-C were observed and the recombinant PERV-A/C were found to be integrated in cells of different organs, but not in the germ line of the animals. Here, the evidence for such in vivo activities of PERVs, including expression as mRNA, protein and virus particles, de novo infection and recombination, will be summarised. These activities make screening of pigs for provirus number and PERV expression level difficult, especially when only blood or ear biopsies are available for analysis. Highly sensitive methods to measure the copy number and the expression level will be required when selecting pigs with low copy number and low expression of PERV as well as when inactivating PERVs using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (CRISPR/Cas) technology.

  • Viruses, Vol. 8, Pages 213: Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro

  • Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, andϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operatorsof N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages.

  • Viruses, Vol. 8, Pages 206: Clomiphene and Its Isomers Block Ebola Virus Particle Entry and Infection with Similar Potency: Potential Therapeutic Implications

  • The 2014 outbreak of Ebola virus (EBOV) in Western Africa highlighted the need for anti-EBOV therapeutics. Clomiphene is a U.S. Food and Drug Administration (FDA)-approved drug that blocks EBOV entry and infection in cells and significantly protects EBOV-challenged mice. As provided, clomiphene is, approximately, a 60:40 mixture of two stereoisomers, enclomiphene and zuclomiphene. The pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which EBOV can persist. Here we compared the ability of clomiphene and its isomers to inhibit EBOV using viral-like particle (VLP) entry and transcription/replication-competent VLP (trVLP) assays. Clomiphene and its isomers inhibited the entry and infection of VLPs and trVLPs with similar potencies. This was demonstrated with VLPs bearing the glycoproteins from three filoviruses (EBOV Mayinga, EBOV Makona, and Marburg virus) and in two cell lines (293T/17 and Vero E6). Visual problems have been noted in EBOV survivors, and viral RNA has been isolated from semen up to nine months post-infection. Since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-EBOV agent, for example, to potentially help ameliorate symptoms in EBOV survivors.

  • Viruses, Vol. 8, Pages 216: Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

  • Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.

  • Viruses, Vol. 8, Pages 209: NXT1, a Novel Influenza A NP Binding Protein, Promotes the Nuclear Export of NP via a CRM1-Dependent Pathway

  • Influenza remains a serious worldwide public health problem. After infection, viral genomic RNA is replicated in the nucleus and packed into viral ribonucleoprotein, which will then be exported to the cytoplasm via a cellular chromosome region maintenance 1 (CRM1)-dependent pathway for further assembly and budding. However, the nuclear export mechanism of influenza virus remains controversial. Here, we identify cellular nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a novel binding partner of nucleoprotein (NP) that stimulates NP-mediated nuclear export via the CRM1-dependent pathway. NXT1-knockdown cells exhibit decreased viral replication kinetics and nuclear accumulated viral RNA and NP. By contrast, NXT1 overexpression promotes nuclear export of NP in a CRM1-dependent manner. Pull-down assays suggest the formation of an NXT1, NP, and CRM1 complex, and demonstrate that NXT1 binds to the C-terminal region of NP. These findings reveal a distinct mechanism for nuclear export of the influenza virus and identify the NXT1/NP interaction as a potential target for antiviral drug development.

  • Viruses, Vol. 8, Pages 210: Measles Virus Host Invasion and Pathogenesis

  • Measles virus is a highly contagious negative strand RNA virus that is transmitted via the respiratory route and causes systemic disease in previously unexposed humans and non-human primates. Measles is characterised by fever and skin rash and usually associated with cough, coryza and conjunctivitis. A hallmark of measles is the transient immune suppression, leading to increased susceptibility to opportunistic infections. At the same time, the disease is paradoxically associated with induction of a robust virus-specific immune response, resulting in lifelong immunity to measles. Identification of CD150 and nectin-4 as cellular receptors for measles virus has led to new perspectives on tropism and pathogenesis. In vivo studies in non-human primates have shown that the virus initially infects CD150+ lymphocytes and dendritic cells, both in circulation and in lymphoid tissues, followed by virus transmission to nectin-4 expressing epithelial cells. The abilities of the virus to cause systemic infection, to transmit to numerous new hosts via droplets or aerosols and to suppress the host immune response for several months or even years after infection make measles a remarkable disease. This review briefly highlights current topics in studies of measles virus host invasion and pathogenesis.

  • Viruses, Vol. 8, Pages 208: Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

  • The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  • Viruses, Vol. 8, Pages 205: The Whitefly Bemisia tabaci Knottin-1 Gene Is Implicated in Regulating the Quantity of Tomato Yellow Leaf Curl Virus Ingested and Transmitted by the Insect

  • The whitefly Bemisia tabaci is a major pest to agricultural crops. It transmits begomoviruses, such as Tomato yellow leaf curl virus (TYLCV), in a circular, persistent fashion. Transcriptome analyses revealed that B. tabaci knottin genes were responsive to various stresses. Upon ingestion of tomato begomoviruses, two of the four knottin genes were upregulated, knot-1 (with the highest expression) and knot-3. In this study, we examined the involvement of B. tabaci knottin genes in relation to TYLCV circulative transmission. Knottins were silenced by feeding whiteflies with knottin dsRNA via detached tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the amounts of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. We suggest that knot-1 protects B. tabaci against deleterious effects caused by TYLCV by limiting the amount of virus associated with the whitefly vector.

  • Viruses, Vol. 8, Pages 207: Local Innate Responses to TLR Ligands in the Chicken Trachea

  • The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV.

  • Viruses, Vol. 8, Pages 204: Inactivation of RNA Viruses by Gamma Irradiation: A Study on Mitigating Factors

  • Effective inactivation of biosafety level 4 (BSL-4) pathogens is vital in order to study these agents safely. Gamma irradiation is a commonly used method for the inactivation of BSL-4 viruses, which among other advantages, facilitates the study of inactivated yet morphologically intact virions. The reported values for susceptibility of viruses to inactivation by gamma irradiation are sometimes inconsistent, likely due to differences in experimental protocols. We analyzed the effects of common sample attributes on the inactivation of a recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein and green fluorescent protein. Using this surrogate virus, we found that sample volume and protein content of the sample modulated viral inactivation by gamma irradiation but that air volume within the sample container and the addition of external disinfectant surrounding the sample did not. These data identify several factors which alter viral susceptibility to inactivation and highlight the usefulness of lower biosafety level surrogate viruses for such studies. Our results underscore the need to validate inactivation protocols of BSL-4 pathogens using“worst-case scenario” procedures to ensure complete sample inactivation.

  • Viruses, Vol. 8, Pages 203: Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures

  • In 2007, the United States– Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethaldose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

  • Viruses, Vol. 8, Pages 202: The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release

  • Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle.

  • Viruses, Vol. 8, Pages 201: Infection Counter: Automated Quantification of in Vitro Virus Replication by Fluorescence Microscopy

  • The ability to accurately and reliably quantify viral infection is essential to basic and translational virology research. Here, we describe a simple and robust automated method for using fluorescence microscopy to estimate the proportion of virally infected cells in a monolayer. We provide details of the automated analysis workflow along with a freely available open-source ImageJ plugin, Infection Counter, for performing image quantification. Using hepatitis C virus (HCV) as an example, we have experimentally verified our method, demonstrating that it is equivalent, if not better, than the established focus-forming assay. Finally, we used Infection Counter to assess the anti-HCV activity of SMBz-CsA, a non-immunosuppressive cyclosporine analogue.

  • Viruses, Vol. 8, Pages 200: A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal

  • RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5′ splice site (5′ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5′ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5′ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.

  • Viruses, Vol. 8, Pages 199: N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription

  • Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as“shut-off”. However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression.

  • Viruses, Vol. 8, Pages 197: Reporter-Expressing, Replicating-Competent Recombinant Arenaviruses

  • Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose an important public health problem in their endemic regions. To date, no Food and Drug Administration (FDA)-licensed vaccines are available to combat human arenavirus infections, and current anti-arenaviral drug therapy is limited to an off-label use of ribavirin that is only partially effective. The development of arenavirus reverse genetic approaches has provided investigators with a novel and powerful approach for the study of arenavirus biology including virus–host interactions underlying arenavirus induced disease. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in arenavirus replication and transcription, as well as particle assembly and budding. Likewise, it is now feasible to rescue infectious arenaviruses containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis. The use of reverse genetics approaches has also allowed the generation of recombinant arenaviruses expressing additional genes of interest. These advances in arenavirus molecular genetics have also facilitated the implementation of novel screens to identify anti-arenaviral drugs, and the development of novel strategies for the generation of arenavirus live-attenuated vaccines. In this review, we will summarize the current knowledge on reporter-expressing, replicating-competent arenaviruses harboring reporter genes in different locations of the viral genome and their use for studying and understanding arenavirus biology and the identification of anti-arenaviral drugs to combat these important human pathogens.

  • Viruses, Vol. 8, Pages 198: KSHV-Mediated Angiogenesis in Tumor Progression

  • Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), is a malignant human oncovirus belonging to the gamma herpesvirus family. HHV-8 is closely linked to the pathogenesis of Kaposi’s sarcoma (KS) and two other B-cell lymphoproliferative diseases: primary effusion lymphoma (PEL) and a plasmablastic variantof multicentric Castleman’s disease (MCD). KS is an invasive tumor of endothelial cells most commonly found in untreated HIV-AIDS or immuno-compromised individuals. KS tumors are highly vascularized and have abnormal, excessive neo-angiogenesis, inflammation, and proliferation of infected endothelial cells. KSHV directly induces angiogenesis in an autocrine and paracrine fashion through a complex interplay of various viral and cellular pro-angiogenic and inflammatory factors. KS is believed to originate due to a combination of KSHV’s efficient strategies for evading host immune systems and several pro-angiogenic and pro-inflammatory stimuli. In addition, KSHV infection of endothelial cells produces a wide array of viral oncoproteins with transforming capabilities that regulate multiple host-signaling pathways involved in the activation of angiogenesis. It is likely that the cellular-signaling pathways of angiogenesis and lymph-angiogenesis modulate the rate of tumorigenesis induction by KSHV. This review summarizes the current knowledge on regulating KSHV-mediated angiogenesis by integrating the findings reported thus far on the roles of host and viral genes in oncogenesis, recent developments in cell-culture/animal-model systems, and various anti-angiogenic therapies for treating KSHV-related lymphoproliferative disorders.

  • Viruses, Vol. 8, Pages 196: Molecular and Genetic Characterization of HIV-1 Tat Exon-1 Gene from Cameroon Shows Conserved Tat HLA-Binding Epitopes: Functional Implications

  • HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores aamp;amp;gt; 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA.

  • Viruses, Vol. 8, Pages 195: Cross-Species Antiviral Activity of Goose Interferons against Duck Plague Virus Is Related to Its Positive Self-Feedback Regulation and Subsequent Interferon Stimulated Genes Induction

  • Interferons are a group of antiviral cytokines acting as the first line of defense in the antiviral immunity. Here, we describe the antiviral activity of goose type I interferon (IFNα) and type II interferon (IFNγ) against duck plague virus (DPV). Recombinant goose IFNα and IFNγ proteins of approximately 20 kDa and 18 kDa, respectively, were expressed. Following DPV-enhanced green fluorescent protein (EGFP) infection of duck embryo fibroblast cells (DEFs) with IFNα and IFNγ pre-treatment, the number of viral gene copies decreased more than 100-fold, with viral titers dropping approximately 100-fold. Compared to the control, DPV-EGFP cell positivity was decreased by goose IFNα and IFNγ at 36 hpi (3.89%; 0.79%) and 48 hpi (17.05%; 5.58%). In accordance with interferon-stimulated genes being the “workhorse” of IFN activity, the expression of duck myxovirus resistance (Mx) and oligoadenylate synthetases-like (OASL) was significantly upregulated (p aamp;amp;lt; 0.001) by IFN treatment for 24 h. Interestingly, duck cells and goose cells showed a similar trendof increased ISG expression after goose IFNα and IFNγ pretreatment. Another interesting observation is that the positive feedback regulation of type I IFN and type II IFN by goose IFNα and IFNγ was confirmed in waterfowl for the first time. These results suggest that the antiviral activities ofgoose IFNα and IFNγ can likely be attributed to the potency with which downstream genes are induced by interferon. These findings will contribute to our understanding of the functional significance of the interferon antiviral system in aquatic birds and to the development of interferon-based prophylactic and therapeutic approaches against viral disease.

  • Viruses, Vol. 8, Pages 194: RNA Encapsidation and Packaging in the Phleboviruses

  • The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts areselectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.

  • Viruses, Vol. 8, Pages 192: Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1

  • The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV) replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses.
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