- Volume 97, Issue 5, 2016

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air–liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

A novel flavivirus, provisionally named Bamaga virus (BgV), was isolated from Culex annulirostris mosquitoes collected from northern Australia. Phylogenetic analysis of the complete nucleotide sequence of the BgV genome revealed it clustered with the yellow fever virus (YFV) group, and was most closely related to Edge Hill virus (EHV), another Australian flavivirus, with 61.9 % nucleotide and 63.7 % amino acid sequence identity. Antigenic analysis of the envelope and pre-membrane proteins of BgV further revealed epitopes common to EHV, dengue and other mosquito-borne flaviviruses. However, in contrast to these viruses, BgV displayed restricted growth in a range of vertebrate cell lines with no or relatively slow replication in inoculated cultures. There was also restricted BgV replication in virus-challenged mice. Our results indicate that BgV is an evolutionary divergent member of the YFV group of flaviviruses, and represents a novel system to study mechanisms of virus host-restriction and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αβ) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

At least two genetically different porcine epidemic diarrhoea virus (PEDV) strains have been identified in the USA: US PEDV prototype and S-INDEL-variant strains. The objective of this study was to compare the pathogenicity differences of the US PEDV prototype and S-INDEL-variant strains in conventional neonatal piglets under experimental infections. Fifty PEDV-negative 5-day-old pigs were divided into five groups of ten pigs each and were inoculated orogastrically with three US PEDV prototype isolates (IN19338/2013, NC35140/2013 and NC49469/2013), an S-INDEL-variant isolate (IL20697/2014), and virus-negative culture medium, respectively, with virus titres of 104 TCID50 ml− 1, 10 ml per pig. All three PEDV prototype isolates tested in this study, regardless of their phylogenetic clades, had similar pathogenicity and caused severe enteric disease in 5-day-old pigs as evidenced by clinical signs, faecal virus shedding, and gross and histopathological lesions. Compared with pigs inoculated with the three US PEDV prototype isolates, pigs inoculated with the S-INDEL-variant isolate had significantly diminished clinical signs, virus shedding in faeces, gross lesions in small intestines, caeca and colons, histopathological lesions in small intestines, and immunohistochemistry staining in ileum. However, the US PEDV prototype and the S-INDEL-variant strains induced similar viraemia levels in inoculated pigs. Whole genome sequences of the PEDV prototype and S-INDEL-variant strains were determined, but the molecular basis of virulence differences between these PEDV strains remains to be elucidated using a reverse genetics approach.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure–activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Although much progress has been made in antiviral agents against hepatitis C virus (HCV) in recent years, novel HCV inhibitors with improved efficacy, optimized treatment duration and more affordable prices are still urgently needed. Here, we report the identification of a natural plant-derived lignan, trachelogenin (TGN), as a potent entry inhibitor of HCV without genotype specificity, and with low cytotoxicity. TGN was extracted and purified from Caulis trachelospermi, a traditional Chinese herb with anti-inflammatory and analgesic effects. A crucial function of TGN was the inhibition of HCV entry during a post-binding step without affecting virus replication, translation, assembly and release. TGN blocked virus infection by interfering with the normal interactions between HCV glycoprotein E2 and the host entry factor CD81, which are key processes for valid virus entry. In addition, TGN diminished HCV cell-to-cell spread and exhibited additional synergistic effects when combined with IFN or telaprevir. In conclusion, this study highlights the effect of a novel HCV entry inhibitor, TGN, which has a target that differs from those of the current antiviral agents. Therefore, TGN is a potential candidate for future cocktail therapies to treat HCV-infected patients.

The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA–RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA–RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific inter-segment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

One of the main research issues regarding infectious pancreatic necrosis virus (IPNV) is its virulence mechanisms. The basis for understanding the molecular virulence determinants of this virus was established over the last decade when it was demonstrated that certain amino acid domains in the VP2 and VP2–NS inter-region determined the level of virulence of IPNV. However, certain variability was still inexplicable and therefore other factors may also be involved. To this end, it was demonstrated recently that infectious bursal disease virus (IBDV), a virus in a different genus of the same family as IPNV, can package more than two dsRNA segments, and that polyploidy may be associated with virulence. In the present report, we analysed the viral fractions obtained after gradient centrifugation to demonstrate that IPNV virions can also package more than two segments, thus indicating that polyploidy is a common birnavirus trait. The differential replication ex vivo and virulence in vivo additionally suggested that such a characteristic is involved in the modulation of virus infectivity. However, although the ex vivo results clearly demonstrated that the replication capacity was enhanced as the viral ploidy increased, the in vivo results could not strongly support a direct relationship between ploidy and virulence to the host, thus suggesting that other virulence determinants are also involved.

Porcine bocavirus (PBoV), a newly identified parvovirus in the family Parvoviridae, has been reported worldwide in swine with post-weaning multisystemic wasting syndrome, respiratory disease or diarrhoea and in asymptomatic swine. NP1 is a protein unique to the genus Bocavirus and its function is not fully understood. In this study, we show that the N-terminal region of PBoV NP1 contains two classical nuclear localization signals (cNLSs) and a non-classical NLS. The N-terminal region also inhibits the promoter activity of IFN-β and IFN-stimulated response element activity the same as full-length NP1 protein, but the PBoV NP1 C-terminal region does not. PBoV NP1 also induces NFκB activation by increasing the phosphorylation of p65, and we demonstrate that the C-terminal region (aa 168–218) is responsible for the induction of NFκB, although the cNLS region of NP1 enhances this activation. The data suggest that PBoV NP1 contains two functionally independent domains in its N- and C-terminal regions. Thus, the N-terminal region of PBoV NP1 is critical for its nuclear localization and IFN-related promoter inhibition, and the C-terminal region is critical for its induction of NFκB.

Felis catus papillomavirus 2 (FcaPV-2) causes premalignant skin lesions in cats and has also been found in a proportion of cutaneous squamous cell carcinomas (SCCs) – a common and potentially fatal cancer of cats. Whilst this could suggest a role of the virus in cancer development, FcaPV-2 has also been detected in skin swabs of normal cats, making it difficult to discern whether the papillomavirus is causing the cancer or merely an ‘innocent bystander’. To distinguish between these two possibilities, real-time PCR was used to determine the viral copy number and the transcriptional activity of FcaPV-2 infections present in 70 formalin-fixed paraffin-embedded skin lesions including 10 papillomavirus-induced premalignant lesions and 60 SCCs. FcaPV-2 gene expression was found in 21 of 60 (35 %) SCCs, all 10 premalignant lesions and none of 10 normal skin samples. The results showed two distinct subsets of SCCs. The majority of the SCCs had low copy numbers of FcaPV-2 DNA (mean of 17 copies per copy of reference gene DNA) and no FcaPV-2 gene expression, suggesting the virus was an incidental finding. In contrast, 20 SCCs had detectable FcaPV-2 E6/E7 gene expression and very high copy numbers of FcaPV-2 DNA, with a mean of 32 930 copies per copy of reference gene DNA. The relative quantity of E6/E7 gene expression and the viral copy number in this group were similar to those found in the papillomavirus-induced premalignant lesions, suggesting that FcaPV-2 may play a role in the development of a subset of feline cutaneous SCCs.

Chronic hepatitis B virus (HBV) infection is a serious public health problem and may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma. It is currently treated with PEGylated IFN-α2a and nucleoside/nucleotide analogues (NAs). However, PEGylated IFN treatment has problems of high cost, low efficiency and side effects. Long-term administration of NAs is necessary to avoid virus relapse, which can cause drug resistance and side effects. New efforts are now being directed to develop novel anti-HBV drugs targeting either additional viral targets other than viral DNA polymerase or host targets to improve the treatment of chronic hepatitis B. In this study, we discovered that oxethazaine, approved for clinic use in a few countries such as Japan, India, South Africa and Brazil, can dose-dependently reduce the levels of HBV envelope antigen, extracellular HBV DNA in supernatants and intracellular HBV total DNA. However, the levels of HBV cccDNA and HBV RNAs were not affected by oxethazaine treatment. Further study confirmed that oxethazaine acts on the virus assembly stage of the HBV life cycle. A study of the mechanisms of oxethazaine suggested that this drug inhibits HBV replication and capsid assembly by blocking the cytosolic calcium‐signalling pathway. Moreover, oxethazaine could inhibit the replication of lamivudine/entecavir-dual-resistant and adefovir-resistant HBV mutants. In conclusion, our study suggests that oxethazaine may serve as a promising drug, or could be used as a starting point for anti-HBV drug discovery.

Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518RRRR521 and 544RLHK547, and that the 28 aa between the two sites were removed after cleavage. Treating infected cells with either convertase or furin inhibitors reduced gB cleavage efficiency. Further, removal of the first furin recognition motif did not affect in vitro growth of EHV-1, while mutation of the second motif greatly affected virus growth. In addition, a second possible signal peptide cleavage site was identified for EHV-1 gB between residues 98 and 99, which was 13 aa downstream of that previously identified.

Antiviral activity of the new chemically synthesized compound NIOCH-14 (a derivative of tricyclodicarboxylic acid) in comparison with ST-246 (the condensed derivative of pyrroledione) was observed in experiments in vitro and in vivo using orthopoxviruses including highly pathogenic ones. After oral administration of NIOCH-14 to outbred ICR mice infected intranasally with 100 % lethal dose of ectromelia virus, it was shown that 50 % effective doses of NIOCH-14 and ST-246 did not significantly differ. The ‘therapeutic window’ varied from 1 day before infection to 6 days post-infection (p.i.) to achieve 100–60 % survival rate. The administration of NIOCH-14 and ST-246 to mice resulted in a significant reduction of ectromelia virus titres in organs examined as compared with the control and also reduced pathological changes in the lungs 6 days p.i. Oral administration of NIOCH-14 and ST-246 to ICR mice and marmots challenged with monkeypox virus as compared with the control resulted in a significant reduction of virus production in the lungs and the proportion of infected mice 7 days p.i. as well as the absence of disease in marmots. Significantly lower proportions of infected mice and virus production levels in the lungs as compared with the control were demonstrated in experiments after oral administration of NIOCH-14 and ST-246 to ICR mice and immunodeficient SCID mice challenged with variola virus 3 and 4 days p.i., respectively. The results obtained suggest good prospects for further study of the chemical compound NIOCH-14 to create a new smallpox drug on its basis.

To elucidate the molecular basis for the rapid oncogenicity of an acutely transforming avian leukosis virus (ALV), isolated from fibrosarcomas in Hy-Line Brown commercial layer chickens infected with ALV subgroup J (ALV-J), the complete genomic structure of the provirus was determined. In addition to ALV-J replication-complete virus SDAU1102, five proviral DNA genomes, named SJ-1, SJ-2, SJ-3, SJ-4 and SJ-5, carrying different lengths of the v-src oncogene were amplified from original tumours and chicken embryo fibroblasts (CEFs) infected with viral stocks. The genomic sequences of the SJ-1–SJ-5 provirus were closely related to that of SDAU1102 but were defective. The results of Western blot analysis and immunohistochemical staining also showed overexpression of the p60v-src protein in infected CEFs and tumour tissue. To the best of our knowledge, this is the first report of the isolation and identification of acutely transforming viruses carrying the v-src oncogene with ALV-J as the helper virus. It also offers insight into the generation of acutely transforming ALVs carrying the v-src oncogene.

Previously, we reported that a new genetically diverse CCR5 (R5) tropic simian/human immunodeficiency virus (SHIV-MK38) adapted to rhesus monkeys became more neutralization resistant to SHIV-infected plasma than did the parental SHIV-KS661 clone. Here, to clarify the significance of the neutralization-resistant phenotype of SHIV in a macaque model, we initially investigated the precise neutralization phenotype of the SHIVs, including SHIV-MK38 molecular clones, using SHIV-MK38-infected plasma, a pooled plasma of human immunodeficiency virus (HIV)-infected individuals, soluble CD4 and anti-HIV-1 neutralizing mAbs, the epitopes of which were known. The results show that SHIV-KS661 had tier 1 neutralization sensitivity, but monkey-adapted R5 tropic SHIV-MK38 acquired neutralization resistance similar to that of tier 2 or 3 as a clone virus. Sequence analysis of the env gene suggested that the neutralization-resistant phenotype of SHIV-MK38 was acquired by conformational changes in Env associated with the net charge and potential N-linked glycosylation sites. To examine the relationship between neutralization phenotype and stably persistent infection in monkeys, we performed in vivo rectal inoculation experiments using a SHIV-MK38 molecular clone. The results showed that one of three rhesus monkeys exhibited durable infection with a plasma viral load of 105 copies ml− 1 despite the high antibody responses that occurred in the host. Whilst further improvements are required in the development of a challenge virus, it will be useful to generate a neutralization-resistant R5 tropic molecular clone of the SHIV-89.6 lineage commonly used for vaccine development – a result that can be used to explore the foundation of AIDS pathogenesis.