- Volume 97, Issue 10, 2016

Genome replication and virion production by most negative-sense RNA viruses (NSVs) occurs exclusively in the cytoplasm, but many NSV-expressed proteins undergo active nucleocytoplasmic trafficking via signals that exploit cellular nuclear transport pathways. Nuclear trafficking has been reported both for NSV accessory proteins (including isoforms of the rabies virus phosphoprotein, and V, W and C proteins of paramyxoviruses) and for structural proteins. Trafficking of the former is thought to enable accessory functions in viral modulation of antiviral responses including the type I IFN system, but the intranuclear roles of structural proteins such as nucleocapsid and matrix proteins, which have critical roles in extranuclear replication and viral assembly, are less clear. Nevertheless, nuclear trafficking of matrix protein has been reported to be critical for efficient production of Nipah virus and Respiratory syncytial virus, and nuclear localization of nucleocapsid protein of several morbilliviruses has been linked to mechanisms of immune evasion. Together, these data point to the nucleus as a significant host interface for viral proteins during infection by NSVs with otherwise cytoplasmic life cycles. Importantly, several lines of evidence now suggest that nuclear trafficking of these proteins may be critical to pathogenesis and thus could provide new targets for vaccine development and antiviral therapies.

Reoviruses (family Reoviridae) infect vertebrate and invertebrate hosts with clinical effects ranging from inapparent to lethal. Here, we describe the discovery and characterization of Largemouth bass reovirus (LMBRV), found during investigation of a mortality event in wild largemouth bass (Micropterus salmoides) in 2015 in WI, USA. LMBRV has spherical virions of approximately 80 nm diameter containing 10 segments of linear dsRNA, aligning it with members of the genus Orthoreovirus, which infect mammals and birds, rather than members of the genus Aquareovirus, which contain 11 segments and infect teleost fishes. LMBRV is only between 24 % and 68 % similar at the amino acid level to its closest relative, Piscine reovirus (PRV), the putative cause of heart and skeletal muscle inflammation of farmed salmon. LMBRV expands the known diversity and host range of its lineage, which suggests that an undiscovered diversity of related pathogenic reoviruses may exist in wild fishes.

Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.

Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.

Non-primate hepacivirus (NPHV), a recently discovered hepatotropic virus infecting horses, is phylogenetically the closest known homologue of hepatitis C virus (HCV). The main route for acquiring HCV infection in childhood is vertical transmission. However, nothing is known about the natural mode of transmission for NPHV. To investigate the possibility of vertically transmitted NPHV infection in horses, 20 Thoroughbred broodmares and their foals were monitored during foaling season 2015 until 6 months post-partum. Prepartal serum was taken from the mares, and during foaling umbilical cord blood and colostrum samples were collected. Postnatal serum samples were taken from the foals after delivery. In addition, serum was taken at 3 and 6 months after foaling from all mares and foals. Samples were analysed for the presence of NPHV RNA by quantitative real-time PCR and for the presence of anti-NPHV NS3 antibodies by luciferase immunoprecipitation system. Identified NPHV isolates were sequenced and phylogenetic analysis of the viral glycoproteins was used to track the course of naturally occurring infections and the circulation of distinct isolates within the herd. At parturition, 16 mares were seropositive, including four viraemic mares. Vertical transmission occurred in one of these four mare–foal pairs. Interestingly, NPHV isolates of newly infected foals and mares after 3 and 6 months cluster in their respective pasture herds suggesting another horizontal route of transmission.

Natural 2′-modified nucleosides are the most widely used antiviral therapy. In their triphosphorylated form, also known as nucleotide analogues, they target the active site of viral polymerases. Viral polymerases have an overall right-handed structure that includes the palm, fingers and thumb domains. These domains are further subdivided into structurally conserved motifs A–G, common to all viral polymerases. The structural motifs encapsulate the allosteric/initiation (N1) and orthosteric/catalytic (N2) nucleotide-binding sites. The current study investigated how nucleotide analogues explore the N2 site of viral polymerases from three genera of the family Flaviviridae using a stochastic, biophysical, Metropolis Monte Carlo-based software. The biophysical simulations showed a statistical distinction in nucleotide-binding energy and exploration between phylogenetically related viral polymerases. This distinction is clearly demonstrated by the respective analogue contacts made with conserved viral polymerase residues, the heterogeneous dynamics of structural motifs, and the orientation of the nucleotide analogues within the N2 site. Being able to simulate what occurs within viral-polymerase-binding sites can prove useful in rational drug designs against viruses.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

Japanese encephalitis virus (JEV) is the main cause of acute viral encephalitis, primarily affecting children and young adults in the Asia-Pacific region. JEV is a vaccine-preventable pathogen, with four types of JE vaccine licensed in different regions of the world. To date, the most common JEV strain used in vaccine development and production is SA14-14-2, an attenuated strain derived from its wild-type parental strain SA14. In this study, we directly compared the phenotypic and genotypic characteristics of SA14 and SA14-14-2 to determine the biological and genetic properties associated with their differential virulence. In susceptible BHK-21 cells, SA14-14-2 grew slightly more slowly and formed smaller plaques than SA14, but unlike SA14, it showed almost no expression of the viral protein NS1′, the product of a conserved predicted RNA pseudoknot-mediated ribosomal frameshift. In weanling ICR mice, SA14-14-2 was highly attenuated in terms of both neuroinvasiveness and neurovirulence, with its median lethal doses invariably over five logs higher than those of SA14 when inoculated intramuscularly and intracerebrally. Interestingly, the neurovirulence of SA14-14-2 was dependent on mouse age, with the 1- to 7-day-old mice being highly susceptible and the 14- to 21-day-old mice becoming resistant to intracerebral inoculation. At the genome level, SA14-14-2 differed from SA14 by 57 nucleotides, including one silent G-to-A substitution at position 3599 within the predicted RNA pseudoknot for NS1′ synthesis; of the 57 differences, 25 resulted in amino acid substitutions. Our data pave the way for the development of new genetically modified JE vaccines.

Favourable genotypes of IFNL3 polymorphism CC for rs12979860 and TT for rs8099917 are strongly associated with the interferon/ribavirin treatment outcome in hepatitis C virus (HCV) patients with genotypes 1 and 4. Contrarily, conflicting results have been reported for patients with HCV genotypes 2 and 3. Therefore, we sought to investigate the association between IFNL3 with sustained virological response (SVR) after treatment to ascertain the predictive value of IFNL3 single-nucleotide polymorphisms (SNPs) in HCV patients with genotype 3. For this purpose, we genotyped five IFNL3 SNPs, rs12980275, rs12979860, rs9109886, rs8099917 and rs7248668, in HCV patients with genotype 3 and assessed its association with SVR, individually and in haplotype. Interestingly, we report that the IFNL3 SNPs we genotyped have shown no association with SVR following treatment, either individually or in haplotype, indicating that genotyping IFNL3 SNPs have limited predictive value in HCV patients with genotype 3. Therefore, we propose that IFNL3 genotyping can be excluded from a patient’s pre-treatment workup for subsequent treatment choice. This will greatly reduce the economic burden for HCV patients with genotype 3 in resource-limited regions, especially South Asia where genotype 3 is predominant.

During human immunodeficiency virus (HIV) infection, soluble CD14 (sCD14) is up-regulated as a consequence of pathological disruption of the gut epithelial barrier, and subsequent increased microbial translocation. Also in hepatitis C virus (HCV)-infected patients with advanced liver fibrosis, increased levels of sCD14 have been reported. Since the liver plays an important role in clearance of translocated bacterial products, hepatic fibrosis may negatively affect clearance and thus contribute to higher sCD14 levels. Chimpanzees (Pan troglodytes) infected with HCV typically show no signs of liver fibrosis. Here, we have tested the hypothesis that increased levels of sCD14 occur in the absence of hepatic fibrosis or microbial translocation in chimpanzees chronically infected with HCV. sCD14 was up-regulated in both HIV/simian immunodeficiency virus (SIV)- and HCV-infected chimpanzees. In HIV/SIV-infected chimpanzees, intestinal fatty acid-binding protein, a marker for gut perturbation, lipopolysaccharide (LPS)-binding-protein and LPS core antibodies, confirm that sCD14 up-regulation was caused by increased microbial translocation. In HCV-infected chimpanzees, no evidence was found for increased microbial translocation despite up-regulation of sCD14. Additionally, the impact of liver fibrosis on microbial translocation was addressed by direct comparison of chimpanzees with a high HCV load and human patients with advanced fibrosis. These data suggest that only in a small minority of HCV patients, hepatic fibrosis corroborates microbial translocation.

We are interested in the influence of nucleotide composition on the fundamental characteristics of the virus RNA genome. Most RNA viruses have genomes with a distinct nucleotide composition, e.g. ranging from minimally 12.9 % to maximally 40.3 % (C- and U-count, respectively, in coronavirus HKU). We present a global analysis of diverse virus types, including plus-strand, minus-strand and double-strand RNA viruses, for the impact of this nucleotide preference on the predicted structure of the RNA genome that is packaged in virion particles and on the codon usage in the viral open reading frames. Several virus-specific features will be described, but also some general conclusions were drawn. Without exception, the virus-specific nucleotide bias was enriched in the unpaired, single-stranded regions of the RNA genome, thus creating an even more striking virus-specific signature. We present a simple mechanism that is based on elementary aspects of RNA structure folding to explain this general trend. In general, the nucleotide bias was the major determinant of the virus-specific codon usages, thus limiting a role for codon selection and translational control. We will discuss molecular and evolutionary scenarios that may be responsible for the diverse nucleotide biases of RNA viruses.

Human noroviruses are the most important viral pathogens causing epidemic acute gastroenteritis, in which the GII.4 viruses have been predominant worldwide for the past decades. During 2014–2015 winter season, a new GII.17 variant emerged as the predominant virus in China surpassing the GII.4 virus in causing significantly increased acute gastroenteritis outbreaks. Genome sequences of the new GII.17 variant was determined and compared with other GII.17 noroviruses, revealing residue substitutions at specific locations, including the histo-blood group antigen-binding site and the putative antigenic epitopes. Further study of GII.17 outbreaks focusing on host susceptibility showed that the new GII.17 variant infected secretor individuals of A, B, O and Lewis types. Accordingly, the P particles of the new GII.17 variant bound secretor saliva samples of A, B, O and Lewis types with significantly higher binding signals than those of the P particles of the previous GII.17 variants. In addition, human sera collected from the outbreaks exhibited stronger blockade against the binding of the new GII.17 P particles to saliva samples than those against the binding between the P particles of previous GII.17 variants and saliva samples. Taken together, our data strongly suggested that the new GII.17 variant gained new histo-blood group antigen-binding ability and antigenic features, which may contribute to its predominance in causing human norovirus epidemics.

One of the most characteristic pathological changes in cats that have succumbed to feline infectious peritonitis (FIP) is a multifocal granulomatous phlebitis. Although it is now well established that leukocyte extravasation elicits the inflammation typically associated with FIP lesions, relatively few studies have aimed at elucidating this key pathogenic event. The upregulation of adhesion molecules on the endothelium is a prerequisite for stable leukocyte–endothelial cell (EC) adhesion that necessarily precedes leukocyte diapedesis. Therefore, the present work focused on the expression of the EC adhesion molecules and possible triggers of EC activation during the development of FIP. Immunofluorescence analysis revealed that the endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was elevated in veins close to granulomatous infiltrates in the renal cortex of FIP patients compared to non-infiltrated regions and specimens from healthy cats. Next, we showed that feline venous ECs become activated when exposed to supernatant from feline infectious peritonitis virus (FIPV)-infected monocytes, as indicated by increased adhesion molecule expression. Active viral replication seemed to be required to induce the EC-stimulating activity in monocytes. Finally, adhesion assays revealed an increased adhesion of naive monocytes to ECs treated with supernatant from FIPV-infected monocytes. Taken together, our results strongly indicate that FIPV activates ECs to increase monocyte adhesion by an indirect route, in which proinflammatory factors released from virus-infected monocytes act as key intermediates.

The viral factors associated with the development of fulminant hepatitis B are not fully understood. We recently found four unique mutations [G to A at nucleotide 1742 (G1742A), C1766T, T1768A and T1809C] in the basal core promoter (BCP) region of a genotype A hepatitis B virus (HBV) strain (FH) obtained from a 53-year-old man with fatal fulminant hepatitis. To elucidate the association of the mutations of the FH genome with the disease, we constructed a 1.3-fold FH genome and its five variants by replacing one or two mutated nucleotides with wild-type nucleotide(s) via site-directed mutagenesis, and transfected human hepatoma cells (HepG2/C3A) with the constructs. There were no discernible differences between FH and two variants (FH_A1742G and FH_C1809T) with regard to viral replication and protein expression. However, in comparison to three other variants (FH_T1766C, FH_A1768T and FH_T1766C/A1768T) with wild-type nucleotide(s) at 1766 and/or 1768, the FH genome exhibited a 2.5–5-fold enhancement of viral replication by heightened pregenomic RNA synthesis and a 1.5–2.5-fold reduction in the hepatitis B e antigen (HBeAg) synthesis by the downregulation of the precore mRNA level. An immunofluorescence analysis revealed the increased and predominant cytoplasmic localization of the core protein in the FH genome. The present study demonstrates that the C1766T/T1768A mutations in the BCP region of genotype A HBV enhance viral replication, downregulate HBeAg expression and are responsible for the predominant localization of the core protein in the cytoplasm, which are likely associated with the development of fulminant hepatitis.

Members of the genus Mastadenovirus including bovine adenovirus 3 (BAdV-3) encode a genus-specific unique protein named pV. The pV encoded by BAdV-3 is a protein of 423 aa showing 40.9 % identity to pV of human adenovirus 2. Here, we report the construction and analysis of recombinant BAdV-3 (BAV.dV) containing deletion of pV. The BAV.dV could only be isolated in CRL.pV cells expressing pV, suggesting that pV appears essential for the infection of BAdV-3. Analysis of BAV.dV suggested that despite affecting some late gene expression in virus-infected cells, there was no significant difference in the incorporation of viral proteins in the mature virions. Moreover, analysis of mature virions revealed degraded capsids leading to change in morphology and infectivity of BAV.dV. Furthermore, analysis of the genome sequence of different clones of BAV.dV passaged in different cell lines revealed no mutations in core proteins pVII and pX\Mu suggesting that the replication defect may not be rescued. Our results suggest that pV is required for proper viral assembly of BAdV-3 as lack of pV produces aberrant capsids. Moreover, altered capsids lead to the production of non-infectious BAV.dV virions.

Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5′ end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.

Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44–50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51–59 was required for the assembly of wild-type levels of infectious virus.