SUMMARY

Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide yet due to the inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo since it establishes persistence in wild type mice, yet causes symptoms resembling gastroenteritis in immune compromised STAT1-/- mice. The lack of reverse genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA based reverse genetics and mouse model system to study persistent MNV-3 infections in wild type (C57BL/6) mice. Viral RNA and infectious virus were detected in the faeces for at least 56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence were detected in the caecum and colon, suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations selected during cell culture passages in RAW264.7 cells that appear in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host, providing new insights into the biology of norovirus infections.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2 %), Phlebotomus longicuspis (30.1 %), Phlebotomus papatasi (12 .0%), Phlebotomus perfiliewi (4.6 %), Phlebotomus langeroni (0.4 %) and Sergentomyia minuta (0.5 %). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. Virus expression is tightly regulated and limited to accessory gene transcripts throughout tumour development. During tumour regression, this regulation is lost and the replication of virus is greatly enhanced. Cultured walleye fibroblasts infected in vitro do not produce significant quantities of infectious virus. Tissue culture cells established by explantation of tumour cells were found to harbour WDSV provirus and to express accessory and structural proteins. The sequence of the provirus showed little variation from a previous WDSV isolate. Retroviral particles were isolated from supernatants from these cells and were able to transfer infection to uninfected walleye fibroblasts. In addition to the virus present in supernatants, much of the virus was cell associated and liberated only by sonication. This virus was found at internal cellular membranes, including mitochondria, and was infectious.

Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55 000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale, we sequenced 62 new isolates and performed an extensive comparative analysis of RABV gene sequence data, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV in non-flying mammals, each of which has a distinct geographical distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we also reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the past 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the global expansion of canid RABV. Overall, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV.

Parvoviruses are small single-stranded DNA viruses that are ubiquitous in nature. Infections with both autonomous and helper-virus dependent parvoviruses are common in both human and animal populations, and many animals are host to a number of different parvoviral species. Despite the epidemiological importance of parvoviruses, the presence and role of genome recombination within or among parvoviral species has not been well characterized. Here we show that natural recombination may be widespread in these viruses. Different genome regions of both porcine parvoviruses and Aleutian mink disease viruses have conflicting phylogenetic histories, providing evidence for recombination within each of these two species. Further, the rodent parvoviruses show complex evolutionary histories for separate genomic regions, suggesting recombination at the interspecies level.

West Nile virus (WNV) is a mosquito-borne flavivirus that was first introduced into the USA in the New York City area in 1999. Since its introduction, WNV has steadily increased both its host and geographical ranges. Outbreaks of the closely related flavivirus, St. Louis encephalitis virus (SLEV), occur in the USA periodically, but levels of activity and host range are more restricted than those of WNV. Understanding the selective pressures that drive arbovirus adaptation and evolution in their disparate mosquito and avian hosts is crucial to predicting their ability to persist and re-emerge. Here, we evaluated the in vivo phenotypes of mosquito cell-adapted WNV and SLEV. Results indicated that in vitro adaptations did not translate to in vivo adaptations for either virus, yet SLEV displayed attenuated growth in both mosquitoes and chickens, while WNV generally did not. In vitro growth analyses also indicated that WNV adaptations could be generalized to cell cultures derived from other mosquito species, while SLEV could not. Analysis of genetic diversity for passaged SLEV revealed a highly homogeneous population that differed significantly from previous results of high levels of diversity in WNV. We hypothesize that this difference in genetic diversity is directly related to the viruses’ success in new and changing environments in the laboratory and that differences in a viruses’ ability to produce and maintain heterogeneous populations in nature may in some instances explain the variable levels of success seen among arboviruses.

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevant in vivo systems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptation in vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage in Culex pipiens mosquitoes. An increase in replicative ability of WNV in C. pipiens was attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previous in vitro studies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged in C. pipiens, moderately attenuated growth in C. pipiens was observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevant in vivo systems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.

West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.

Summary

We have previously shown that OTK18, a human immunodeficiency virus (HIV)-inducible zinc finger protein, reduces progeny virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 viral long terminal repeat (LTR) promoter activity. Through the use of LTR scanning mutant vectors, we have defined the specific regions responsible for OTK18-mediated LTR suppression. Two different LTR regions were identified as potential OTK18 binding sites by an enhanced DNA-transcription factor ELISA system; the negative regulatory element (NRE) at -255 / −238 and Ets binding site (EBS) at −150 / −139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18-antiretroviral activity in human macrophages.

SUMMARY

Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The nonstructural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural-gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

Summary

When Ebola virus nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 amino acid residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 amino acids. These findings enhance our knowledge of Ebola virus assembly and understanding of the Ebola virus life cycle.

Summary

A/Hong Kong/213/97 (HK213; H5N1), isolated from a human, binds to both avian- and human-type receptors due to an HA mutation likely acquired during adaptation to humans. Duck passage of this virus conferred lethality in ducks. Sequence analyses of the duck-passaged virus revealed that its HA gene reverted back to one recognizing only avian-type receptors, and consequently it bound human tissue to a lesser extent. This finding suggests that viruses with human-type receptor specificity are unlikely to be maintained in waterfowl, unlike those with the human-type PB2 mutation, such as H5N1 viruses of the Qinghai lineage.

When Ebola virus nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA, which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 aa residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 aa. These findings enhance our knowledge of Ebola virus assembly and understanding of the Ebola virus life cycle.

A/Hong Kong/213/97 (HK213; H5N1), isolated from a human, binds to both avian- and human-type receptors, due to a haemagglutinin (HA) mutation probably acquired during adaptation to humans. Duck passage of this virus conferred lethality in ducks. Sequence analyses of the duck-passaged virus revealed that its HA gene reverted back to one recognizing only avian-type receptors, and consequently it bound human tissue to a lesser extent. This finding suggests that viruses with human-type receptor specificity are unlikely to be maintained in waterfowl, unlike those with the human-type PB2 mutation, such as H5N1 viruses of the Qinghai Lake lineage.

Summary

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is closely related to vesicular stomatitis virus (VSV), a prototype of rhabdoviruses. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA: GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5’-monophosphorylated viral mRNA-start sequence to GDP to produce a capped RNA, and the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA- and leader RNA-start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme-pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active site HR motif for the PRNTase reaction at the step of the enzyme-pRNA intermediate formation.

SUMMARY

Recent large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA, and NP of human influenza isolates (‘human-like amino acids’) in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA : GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5′-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme–pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme–pRNA intermediate formation.

Recent large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA and NP proteins of human influenza isolates (‘human-like amino acids’) in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

The human antibody response to polyomavirus capsid proteins is not well characterized. Recombinant BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40) virus-like particles (VLP) were produced in a baculovirus system, and mouse monoclonal antibodies (mAbs) to these proteins were generated using standard methods. Nine of 12 BKV mAbs showed neutralizing activity. The non-neutralizing antibodies also bound BKV pseudocapsids in an ELISA binding assay. Most antibodies recognized conformational species-specific epitopes, but several exceptions were found: (i) BKV mAb BK-F11 cross-reacted with a linear buried epitope common to both JCV and SV40 pseudocapsids, (ii) two of six JCV antibodies (JC-6.7 and JC-7.9) and two of 13 SV40 antibodies (VP1-H2 and VP1-I2) recognized linear buried epitopes common to all three viruses and (iii) SV40 antibody VP1-E5 recognized a linear surface epitope on JCV pseudocapsids.

Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor α and alpha/beta interferon (IFN-α/β). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4+ T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor α and alpha/beta interferon (IFN-α/β). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4+ T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

Diarrhea remains a significant cause of morbidity and mortality in developing countries where numerous cases remain without identified etiology. Astroviruses are a recently identified cause of animal gastroenteritis which currently includes two species suspected of causing human diarrhea. Using a combination of viral metagenomics and pan-astrovirus RT-PCR we analyzed human stools samples from different continents for astrovirus related RNA sequences. We identified variants of the two known human astrovirus species plus, based on genetic distance criteria, three novel astrovirus species all distantly related to mink and ovine astroviruses which we provisionally named HMOAstV species A through C. A complete genome of species A displayed all the conserved characteristics of mammalian astroviruses. Each of the now three groups of astroviruses found in human stool (HAstV, AstV-MLB, HMOAstV) being more closely related to animal astroviruses than to each other indicates that human astroviruses may periodically emerge from zoonotic transmissions. Based on the pathogenic impact of their closest phylogenetic relatives in animal further investigations of the role of HMOAstV, so far detected in Nigeria, Nepal and Pakistan, in human gastroenteritis are warranted.

Diarrhoea remains a significant cause of morbidity and mortality in developing countries where numerous cases remain without identified aetiology. Astroviruses are a recently identified cause of animal gastroenteritis which currently includes two species suspected of causing human diarrhoea. Using pan-astrovirus RT-PCR, we analysed human stool samples from different continents for astrovirus-related RNA sequences. We identified variants of the two known human astrovirus species plus, based on genetic distance criteria, three novel astrovirus species all distantly related to mink and ovine astroviruses, which we provisionally named HMOAstV species A–C. The complete genome of species A displayed all the conserved characteristics of mammalian astroviruses. Each of the now three groups of astroviruses found in human stool (HAstV, AstV-MLB and HMOAstV) were more closely related to animal astroviruses than to each other, indicating that human astroviruses may periodically emerge from zoonotic transmissions. Based on the pathogenic impact of their closest phylogenetic relatives in animals, further investigations of the role of HMOAstV, so far detected in Nigeria, Nepal and Pakistan, in human gastroenteritis are warranted.