- Volume 94, Issue 10, 2013

Junin virus (JUNV) has been identified as the aetiological agent of Argentine haemorrhagic fever (AHF), which is a serious public health problem with approximately 5 million people at risk. It is treated as a potential bioterrorism agent because of its rapid transmission by aerosols. JUNV is a negative-sense ssRNA virus that belongs to the genus Arenavirus within the family Arenaviridae, and its genomic RNA contains two segments encoding four proteins. Among these, the nucleoprotein (NP) has essential roles in viral RNA synthesis and immune suppression, but the molecular mechanisms of its actions are only partially understood. Here, we determined a 2.2 Å crystal structure of the C-terminal domain of JUNV NP. This structure showed high similarity to the Lassa fever virus (LASV) NP C-terminal domain. However, both the structure and function of JUNV NP showed differences compared with LASV NP. This study extends our structural insight into the negative-sense ssRNA virus NPs.

We describe the discovery and characterization of a novel paramyxovirus, porcine parainfluenza virus 1 (PPIV-1), from swine. The virus was detected in 12 (3.1 %) of 386 nasopharyngeal and two (0.7 %) of 303 rectal swab samples from 386 deceased pigs by reverse transcription-PCR, with viral loads of up to 106 copies ml−1. Complete genome sequencing and phylogenetic analysis showed that PPIV-1 represented a novel paramyxovirus within the genus Respirovirus, being most closely related to human parainfluenza virus 1 (HPIV-1) and Sendai virus (SeV). In contrast to HPIV-1, PPIV-1 possessed a mRNA editing function in the phosphoprotein gene. Moreover, PPIV-1 was unique among respiroviruses in having two G residues instead of three to five G residues following the A6 run at the editing site. Nevertheless, PPIV-1, HPIV-1 and SeV share common genomic features and may belong to a separate group within the genus Respirovirus. The presence of PPIV-1 in mainly respiratory samples suggests a possible association with respiratory disease, similar to HPIV-1 and SeV.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime–boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309–320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime–boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

To investigate the potential role of non-human primates (NHPs) in a dengue virus (DENV) epidemic, we conducted serological and genomic studies using plasma samples collected from 100 cynomolgus monkeys (Macaca fascicularis) in an animal breeding facility in the Philippines. An ELISA revealed 21 monkeys with a positive IgM reaction and 19 positive for IgG. Five of the monkeys were positive for both IgM and IgG. Of the 21 IgM-positive samples, a neutralization assay identified seven containing DENV-specific antibodies. We amplified the viral non-structural 1 (NS1) gene in two and the envelope (E) gene in one of these seven samples by RT-PCR. Phylogenetic analyses revealed that these DENV genes belonged to the epidemic DENV-2 family, not the sylvatic DENV family. These results suggest that NHPs may serve as a reservoir of epidemic DENV; therefore, the ecology of the urban DENV infection cycle should be investigated in these animals in detail.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

Dengue virus (DENV) infection affects millions of people annually and has the potential to cause fatal haemorrhagic fever and shock. Although the underlying pathogenesis of severe dengue illness is still unclear, current evidence suggests that severe disease progression has an immunological basis. In this study, we investigated the role of caspase-1 during host–pathogen interactions within DENV-infected human monocytes. Using DENV-infected primary monocytes, we examined caspase-1 at various levels of gene expression and probed for potential immune consequences mediated by caspase-1 such as secretion of pro-inflammatory IL-1β and pyroptotic cell death. We report that DENV-infected monocytes upregulated functional caspase-1 mRNA and pro-caspase-1 activation as a late response to infection. In addition, we found that caspase-1 is responsible for IL-1β secretion and pyroptosis of DENV-infected monocytes. Together, our results show that late caspase-1 activation within DENV-infected monocytes can contribute to pro-inflammatory outcomes that might play a role in dengue immunopathogenesis.

Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinants produced infectivity titres of 102.5–104.5 f.f.u. ml−1. Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein–protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe and effective vaccine is sought urgently. As part of a programme to generate a designed highly attenuated vaccine, we report here the recovery of AHSV from a complete set of RNA transcripts synthesized in vitro from cDNA clones. We have demonstrated the generation of mutant and reassortant AHSV genomes, their recovery, stable passage, and characterization. Our findings provide a new approach to investigate AHSV replication, to design AHSV vaccines and to aid diagnosis.

A novel fish reovirus, Hubei grass carp disease reovirus (HGDRV; formerly grass carp reovirus strain 104, GCRV104), was isolated from diseased grass carp in China in 2009 and the full genome sequence was determined. This reovirus was propagated in a grass carp kidney cell line with a typical cytopathic effect. The total size of the genome was 23 706 bp with a 51 mol% G+C content, and the 11 dsRNA segments encoded 12 proteins (two proteins encoded by segment 11). A nucleotide sequence similarity search using blastn found no significant matches except for segment 2, which partially matched that of the RNA-dependent RNA polymerase (RdRp) from several viruses in the genera Aquareovirus and Orthoreovirus of the family Reoviridae. At the amino acid level, seven segments (Seg-1 to Seg-6, and Seg-8) matched with species in the genera Aquareovirus (15–46 % identities) and Orthoreovirus (12–44 % identities), while for four segments (Seg-7, Seg-9, Seg-10 and Seg-11) no similarities in these genera were found. Conserved terminal sequences, 5′-GAAUU----UCAUC-3′, were found in each HGDRV segment at the 5′ and 3′ ends, and the 5′-terminal nucleotides were different from any known species in the genus Aquareovirus. Phylogenetic analysis based on RdRp amino acid sequences from members of the family Reoviridae showed that HGDRV clustered with aquareoviruses prior to joining a branch common with orthoreoviruses. Based on these observations, we propose that HGDRV is a new species in the genus Aquareovirus that is distantly related to any known species within this genus.

Avian leukosis virus (ALV) is the most common oncogenetic retrovirus that emerges spontaneously as a result of recombination between exogenous viruses, exogenous viruses and endogenous viruses, and exogenous viruses and non-homologous cellular genes. In the present study, two natural recombinant avian leukosis viruses (rALVs) (LC110515-5 and LC110803-5) carrying a subgroup C gp85 gene, a subgroup E gp37 gene, and a subgroup J 3′UTR and 3′LTR were isolated from two different lines of layer flocks, Black-bone silky fowl (BSF) and commercial layer chicken, that suffered from myeloid leukosis. Although tumours were not observed in rALV-infected individual chickens, other non-neoplastic inflammatory lesions were evident. The two rALVs were cultured on DF-1 cells and identified by PCR, immunofluorescence assay and gene sequencing. The gp85 nucleotide sequence in the two isolates displayed a high identity (>95 %) with that of the gp85 gene in ALV-C, but the identity was less than 90 % with ALV-A/B/D/E and only 51 % with ALV-J. Phylogenetic analysis of the nucleotide and amino acid sequences confirmed that the two isolates were recombinant between ALV-C, ALV-E and ALV-J. Subgroup C ALV is rarely found in field cases. This report is the first to provide evidence that ALV-C has recombined with ALV-E and ALV-J in two different chicken lines. The source and characteristics of the two rALVs and ALV-C need to be further investigated.

MicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs that modulate gene expression at the post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have demonstrated that a number of cellular miRNAs play a role during viral infection. In this study, a luciferase-reporter system carrying the 5′ untranslated region (5′ UTR) and 3′ UTR of avian leukosis virus subgroup J (ALV-J) was used to determine whether cellular miRNAs are involved in ALV-J infection. The miRNA gga-miR-1650 was screened for its potential interaction with the 5′ UTR of ALV-J and the ability to suppress luciferase-reporter activity. A mutational analysis of predicted gga-miR-1650-binding sites showed that the 5′ and 3′ ends of gga-miR-1650 contributed to the interaction between gga-miR-1650 and its target located at the 5′ UTR. Overexpression of miRNA gga-miR-1650 was shown to downregulate the expression of the Gag protein and influence the replication of ALV-J through binding to the 5′ UTR. Overall, this report provides the basis for the development of new strategies for anti-ALV-J intervention.

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

Recent studies suggest that low levels of reactive oxygen species (ROS) often modulate normal intracellular signalling pathways, determine cell fates and control cell proliferation. We found that infection of astrocytes with the neuropathogenic retrovirus ts1, a mutant of Moloney murine leukemia retrovirus, upregulated ROS at low levels during the early phase of infection. This upregulation of intracellular ROS with downregulation of NADPH levels during the early phase of ts1 infection was a separate event from the upregulation of ROS during the late phase while ts1-mediated cell death occurred. The treatment of apocynin, a potential inhibitor of NADPH oxidase (NOX), inhibited establishment of the ts1 virus in the host cell. These results suggested that ROS generated as a consequence of the activation of NOX may play an important role in the early events of the virus life cycle leading to the establishment of the virus in the host cell. The in vitro results were further supported by an in vivo experiment which showed that the treatment of apocynin decreased viral titre in the ts1-infected mouse brain and increased the lifespan of infected mice. This study provides the first in vitro and in vivo evidence on a mechanism for how ROS are involved in ts1 retrovirus infection and ts1-mediated neurodegenerative disease. Our findings focusing on the early phase of the ts1 retrovirus life cycle could provide a better understanding of retroviral life cycle, which may offer specific therapeutic targets for suppressing viral replication and alleviating neurodegenerative symptoms in a mouse model.

Ten Hepatitis B virus (HBV) genotypes, as well as numerous subgenotypes, have been described in well-characterized ethnogeographical populations. Martinique has been at a crossroads between Africa, Europe, India and the Americas because of the slave trade (17th–19th centuries), followed by an important immigration of Indian and West African workers. In this work, we aimed to study the molecular epidemiology of HBV infection in Martinique according to this unique settlement pattern. To that end, blood samples from 86 consecutive HBV-infected patients from the main hospitals of the island, were retrospectively analysed. Direct sequencing of the pre-S1 or pre-C-C region or complete genome sequencing, followed by phylogenetic analyses were performed. HBV genotypes were: HBV/A1 (68.6 %), HBV/A2 (10.5 %), HBV/D, mainly HBV/D3 and HBV/D4 (8.1 %), HBV/F (3.5 %), and also HBV/E (2.3 %), two strains isolated from two West-African patients. Moreover, 74 % of the HBeAg-negative strains harboured classical pre-C-C mutations, and most HBV/A1 strains also containing specific mutations. Finally, various patterns of deletion mutants in pre-S and pre-C-C regions were found. In conclusion, our findings point to historical and migration-related issues in HBV-genotype distribution suggesting that HBV/A1, but not HBV/E, was imported from Africa during the slave trade, and further supporting the hypothesis that HBV/E has emerged recently in West Africa (<150 years). Potential origins of ‘European’ HBV/A2 and HBV/D3, ‘Amerindian’ HBV/F, and HBV/D4 strains are also discussed. Such HBV genetic diversity, beyond its epidemiological interest, may have a clinical impact on the natural history of HBV infection in Martinique.

To elucidate the spatiotemporal phylodynamics, dispersion and evolutionary processes underlying the emergence of novel porcine parvovirus 2 (PPV2), PPV3 and PPV4 species, we analysed all available complete capsid genes, together with ours, obtained in Europe. Bayesian phylogeography indicates that Romania (PPV2 and PPV4) and Croatia (PPV3) are the most likely ancestral areas from which PPVs have subsequently spread to other European countries and regions. The timescale of our reconstruction supported a relatively recent history of the currently circulating novel PPV species (1920s to 1980s) in the domestic or sylvatic host. While PPV2 strains exhibited a large genetic exchange characterized by significant recombination and gene flow between distinct regions and hosts, PPV3 and PPV4 showed a diversification reflected by the accumulation of geographically structured polymorphisms. The RNA-like evolutionary rates detected inter- and intrahost recombination and the positive selection sites provided evidence that the PPV2–4 capsid gene plays a prominent role in host adaptation.

Thirteen novel avipoxviruses were isolated from birds from different regions of South Africa. These viruses could be divided into six groups, according to gross pathology and pock appearance on chick chorioallantoic membranes (CAMs). Histopathology revealed distinct differences in epidermal and mesodermal cell proliferation, as well as immune cell infiltration, caused by the different avipoxviruses, even within groups of viruses causing similar CAM gross pathology. In order to determine the genetic relationships among the viruses, several conserved poxvirus genetic regions, corresponding to vaccinia virus (VACV) A3L (fpv167 locus, VACV P4b), G8R (fpv126 locus, VLTF-1), H3L (fpv140 locus, VACV H3L) and A11R–A12L (fpv175–176 locus) were analysed phylogenetically. The South African avipoxvirus isolates in this study all grouped in clade A, in either subclade A2 or A3 of the genus Avipoxvirus and differ from the commercial fowlpox vaccines (subclade A1) in use in the South African poultry industry. Analysis of different loci resulted in different branching patterns. There was no correlation between gross morphology, histopathology, pock morphology and phylogenetic grouping. There was also no correlation between geographical distribution and virus phenotype or genotype.