- Volume 89, Issue 8, 2008

To date, all studies regarding hepatitis C virus (HCV) F protein have been based on expression in vitro/in vivo of recombinant protein or monoclonal antibodies derived from genotype 1a or 1b sequences, but not from other genotypes. The objective of this study was to prepare a putative genotype 2 recombinant F protein and evaluate its reactivity in plasma from individuals with chronic HCV infection or who had recovered from infection. One genotype 2 strain was selected for F protein (F-2) and core expression in bacterial culture. An ELISA was developed and applied to samples from patients with chronic infection or recovered infection of various genotypes. The anti-F-2 response in 117 samples showed a significantly higher reactivity in chronic than in recovered HCV-infected blood donors (P<0.001), but no difference was found among genotypes. However, the correlation between anti-F and anti-core was more significant in genotypes 1 and 2 than in genotype 3. Anti-F-2 titres were also significantly higher in chronic than in recovered individuals (P<0.0001). Antibody titres to recombinant genotype 2 core protein or to genotype 1 multiple proteins used in commercial anti-HCV assays paralleled the anti-F-2 end-point antibody titre. This study thus demonstrated the antigenicity of genotype 2 HCV F protein, although the exact location of the natural frameshift position remains unknown. The difference in anti-F-2 response between chronic and recovered infection, the cross-reactivity irrespective of genotype and the correlation of antibody response with structural and non-structural antigens suggest that the immune response to F protein is an integral part of the natural HCV infection.

St. Louis encephalitis virus (SLEV) has been regularly isolated throughout the Americas since 1933. Previous phylogenetic studies involving 62 isolates have defined seven major lineages (I–VII), further divided into 14 clades. In this study, 28 strains isolated in Texas in 1991 and 2001–2003, and three older, previously unsequenced strains from Jamaica and California were sequenced over the envelope protein gene. The inclusion of these new sequences, and others published since 2001, has allowed better delineation of the previously published SLEV lineages, in particular the clades of lineage II. Phylogenetic analysis of 106 isolates identified 13 clades. All 1991 and 2001–2003 isolates from Nueces, Jefferson and Harris Counties (Texas Gulf Coast) group in clade IIB with other isolates from these counties isolated during the 1980s and 1990s. This lack of evidence for introduction of novel strains into the Texas Gulf Coast over a long period of time is consistent with overwintering of SLEV in this region. Two El Paso isolates, both from 2002, group in clade VA with recent Californian isolates from 1998–2001 and some South American strains with a broad temporal range. Overall, these data are consistent with multiple introductions of SLEV from South America into North America, and provide support for the hypothesis that in most situations, SLEV circulates within a locality, with occasional incursions from other areas. Finally, SLEV has much lower nucleotide (10.1 %) and amino acid variation (2.8 %) than other members of the Japanese encephalitis virus complex (maximum variation 24.6 % nucleotide and 11.8 % amino acid).

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras–Erk signalling, however GBV-B NS5A expression did result in the elevation of β-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras–Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.

Analysis of complete polyprotein-encoding sequences of hepatitis C virus genotype 1b (HCV-1b) showed evidence not only of past purifying selection but also of abundant slightly deleterious non-synonymous variants subject to ongoing purifying selection. The NS3 protein (with protease and NTPase/helicase activity) revealed less evidence of purifying selection acting on the cytotoxic T cells (CTL) epitopes than did the other proteins, whereas outside the CTL epitopes NS3 was more conserved than the other proteins. Moreover, NS3 showed a high incidence of forward-and-backward or parallel non-synonymous changes in CTL epitopes, as measured by the consistency index across the phylogeny of HCV-1b genomes computed at non-singleton non-synonymous polymorphic sites. This result implies that certain non-synonymous mutations have recurred frequently throughout the phylogeny in the codons encoding the epitopes in NS3. This pattern is most easily explained by the frequent re-occurrence of the same set of escape mutations in CTL epitopes of NS3, which are selectively favoured within hosts expressing the presenting class I major histocompatibility complex molecule, but are subject to purifying selection at the population level. The fact that this pattern is most strikingly observed in the case of NS3 suggests that the evolutionary conflict between immune escape and functional constraint on the protein is more acute in the case of NS3 than any of the other proteins of HCV-1b.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, replicates primarily at the endoplasmic reticulum and thereby triggers apoptosis of infected cells. This study investigated the hierarchical activation of the caspase network induced by JEV infection. It was found that JEV activated the initiators caspase-8 and -9, as well as effector caspase-3, in infected baby hamster kidney and mouse neuroblastoma (N18) cells. In neuronal N18 cells, JEV infection triggered cytochrome c release from mitochondria, which in turn activated caspase-9 and -3. Treatment of JEV-infected N18 cells with cyclosporin A or ruthenium red, which attenuate mitochondrial injuries, blocked activation of caspase-9 or -3, typifying that, in neuronal cells, this apoptosis involves the mitochondrial pathway. Alternatively, in caspase-3-deficient MCF-7 cells, JEV persisted and readily triggered a typical apoptotic response, including cytochrome c release and full activation of caspase-9 and -8 along with caspase-6, indicating that JEV did not require caspase-3 to manifest caspase-8 activation and apoptosis. Interestingly, a Fas-associated death-domain-containing protein (FADD) dominant-negative mutant, which interfered with transmission of the extracellular death signals into cells through the Fas/tumour necrosis factor (TNF) receptor, failed to block JEV-induced apoptosis and caspase-8 activation, implying that receptor oligomerization of the Fas/TNF pathway might not participate in JEV-induced apoptosis. Taken together, these results illustrate that JEV infection triggers caspase cascades involving the initiators caspase-8 and -9, probably through FADD-independent but mitochondrion-dependent pathways.

Previously, we have shown that mice defective in granule exocytosis and/or Fas.L/Fas-mediated cytolytic pathways are significantly more resistant to alphavirus, Semliki Forest virus (SFV), infection compared with wild-type mice. Here, we evaluated SFV replication in different tissues of mice defective in both cytolytic pathways (perf−/−xgld) relative to that in wild-type counterparts and found that viral replication in perf−/−xgld mice is remarkably restricted. Although the mechanism responsible for this observation is yet to be established, the lower virus titres found in these mice indicate that the role of cytolytic effector molecules in antiviral immunity needs to be re-evaluated.

Kerala State in India was gripped by a renewed and widespread outbreak of Chikungunya virus (CHIKV) infection during 2007. Here, we report the A226V mutation in the glycoprotein envelope 1 (E1) gene of the virus among isolates collected from the three worst-affected districts of the state during this outbreak. This mutation had already been suggested to be directly responsible for a significant increase in CHIKV infectivity in Aedes albopictus. The badly affected districts in Kerala State during 2007 have abundant rubber plantations, which supported prolific breeding of Ae. albopictus mosquitoes. The abundance of Ae. albopictus in the region and molecular evolution of CHIKV may be contributing factors for the renewed epidemic of chikungunya fever during 2007.

An enterovirus strain (designated D207) isolated from a Slovakian diabetic child and originally serotyped as coxsackievirus A9 (CAV-9) was found to cause rapid cytolysis coinciding with severe functional damage of the surviving cells in primary cultures of human pancreatic islets. This finding prompted us to clone the isolate for full-length genome sequencing and molecular characterization as the prototype strain of CAV-9 is known to cause only minimal damage to insulin-producing β-cells. Based on capsid-coding sequence comparisons, the isolate turned out to be echovirus 11 (E-11). Phylogenetic analyses demonstrated that E-11/D207 was closely related to a specific subgroup B of E-11 strains known to cause uveitis. To study further antigenic properties of isolate E-11/D207 and uveitis-causing E-11 strains, neutralization experiments were carried out with CAV-9- and E-11-specific antisera. Unlike the prototype strains, the isolate E-11/D207 and uveitis-causing E-11 strains were well neutralized with both CAV-9- and E-11-specific antisera. Attempts to identify recombination of the capsid coding sequences as a reason for double-reactivity using the Simplot analysis failed to reveal major transferred motifs. However, peptide scanning technique was able to identify antigenic regions of capsid proteins of E-11/D207 as well as regions cross-reacting with an antiserum raised to CAV-9. Thus, double specificity of E-11/D207 seems to be a real characteristic shared by the phylogenetically closely related virus strains in the genetic subgroup B of E-11.

The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis.

Human metapneumovirus (hMPV) strains are classified into two genetic groups, A and B, each of which is further divided in two genetic subgroups, A1, A2, B1 and B2. hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. Although the amino acid sequences of hMPV F protein are highly conserved, those of the G protein are highly variable with low amino acid identity between the two groups. To address the antigenic variation between the genetic subgroups, we developed an immunofluorescence assay (IFA) method using Trichoplusia ni (Tn5) insect cells infected with each recombinant baculovirus-expressed hMPV G (Bac-G) protein of the four genetic subgroups. The titre of each antibody to the four Bac-G proteins was measured by the IFA in 12 paired serum samples obtained from children infected with hMPV of each genetic subgroup. Although 11 of the 12 acute-phase serum samples in paired samples were negative for the antibody to any Bac-G proteins, all of the convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Since the antibody response to hMPV G protein was transient and genetic subgroup-specific without cross-reactivity, four genetic subgroups on the basis of hMPV G protein could be identified as different serotypes. This assay may be useful for the study of immune responses of humans to different hMPV strains, especially for clarifying the risk of reinfection with hMPV.

Human metapneumovirus (HMPV) is a recently discovered pathogen that causes a significant proportion of respiratory infections in young infants, the elderly and immunocompromised patients. Very little is known regarding the cellular signalling elicited by this virus in airway epithelial cells, the target of HMPV infection. In this study, we investigated the role of the RNA helicases retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5) as the main pattern recognition receptors (PRRs) involved in viral detection and subsequent expression of proinflammatory and antiviral genes. HMPV infection readily induced RIG-I and MDA-5 gene and protein expression in A549 cells, a type II-like alveolar epithelial cell line. Expression of dominant-negative (DN) RIG-I or downregulation of RIG-I gene expression using small interfering RNA (siRNA) significantly decreased HMPV-induced beta interferon (IFN-β), interleukin (IL)-8 and RANTES gene transcription, by inhibiting viral-induced activation of nuclear factor (NF)-κB and interferon regulatory factor (IRF), leading to enhanced viral replication. On the other hand, MDA-5 did not seem to play a significant role in HMPV-induced cellular responses. Mitochondrial antiviral signalling protein (MAVS), an adaptor protein linking both RIG-I and MDA-5 to downstream activation of IRF-3 and NF-κB, was also necessary for HMPV-induced cellular signalling. Expression of a DN MAVS significantly reduced IFN-β and chemokine gene transcription, by inhibiting NF-κB- and IRF-dependent gene transcription, in response to HMPV infection. Our results show that HMPV activates the RIG-I–MAVS signalling pathway in airway epithelial cells, leading to the expression of important proinflammatory and antiviral molecules involved in the innate immune response to viruses.

To gain further insight into the molecular epidemiology of Hantaan virus (HTNV) in Guizhou, China, rodents were captured in this region in 2004 and 2005. In addition, serum samples were collected from four patients. Ten hantaviruses were isolated successfully in cell culture from four humans, two Apodemus agrarius, three Rattus norvegicus and one Rattus nitidus. The nucleotide sequences for their small (S), medium (M) and partial large (L) segments were determined. Phylogenetic analysis of the S and M segment sequences revealed that all of these isolates belong to the species HTNV, suggesting a spillover of HTNV from A. agrarius to Rattus rats. All available isolates from Guizhou were divided into four distinct groups either in the S segment tree or in the M segment tree. The clustering pattern of these isolates in the S segment tree was not in agreement with that in the M or L segment tree, showing that genetic reassortment between HTNV had occurred naturally. Analysis of the S segment sequences from available HTNV strains indicated that they formed three clades. The first clade, which comprised only viruses from Guizhou, was the outgroup of clades II and III. The viruses in the second clade were found in Guizhou and mainly in the far-east Asian region, including China. However, the viruses in the third clade were found in most areas of China, including Guizhou, in which haemorrhagic fever with renal syndrome (HFRS) is endemic. Our results reveal that the highest genetic diversity of HTNV is in a limited geographical region of Guizhou, and suggest that Guizhou might be a radiation centre of the present form of HTNV.

The aim of this study was to determine the susceptibility of insectivorous bats (using the big brown bat as a model) to infection with European bat lyssavirus type 1a (EBLV-1a), to assess the dynamics of host immune responses and to evaluate the opportunity for horizontal viral transmission within colonies. Two isolates of EBLV-1a, originating from Slovakia (EBLV-1aSK) and Germany (EBLV-1aGE), were tested. Four different routes of inoculation were used with isolate EBLV-1aSK [104.8 mouse intracerebral median lethal dose (MICLD50) in 50 μl]: intramuscular (i.m.) in the deltoid area or masseter region, per os (p.o.) and intradermal (i.d.) scratches. Isolate EBLV-1aGE (103.2 and 102.2 MICLD50 in 20 μl) was inoculated via the intranasal (i.n.), i.m. (low- and high-dose groups, into pectoral muscles); p.o. and intracerebral (i.c.) routes. None of the bats infected by the i.n., p.o. or i.d. route with either virus isolate developed disease during the experiments (91 or 120 days, respectively). Incubation periods were 9–12 days for i.c.-inoculated bats (66 % mortality), 12–33 days for bats inoculated i.m. with the higher dose (23–50 % mortality) and 21–58 days in bats inoculated i.m. with the lower dose of virus (57 % mortality). Virus or viral RNA in bat saliva was detected occasionally, as early as 37 days before death. All i.d.-inoculated and the majority of i.m.-inoculated bats seroconverted within 7–10 days of inoculation. These observations suggest that exposure of bats to varying doses of EBLV-1 from rabid conspecifics via natural (i.d.) routes could lead to an abortive infection and serve as a natural mode of immunization resulting in the presence of virus-neutralizing antibodies in free-ranging bats.

The identification and characterization of a functional cellular receptor for equine infectious anemia virus (EIAV), designated equine lentivirus receptor-1 (ELR1), a member of the tumour necrosis factor receptor protein family, has been reported previously [ Zhang, B. et al. (2005). Proc Natl Acad Sci U S A, 102 , 9918–9923 ]. The finding of a single receptor for EIAV is distinct from feline, simian and human immunodeficiency viruses, which typically utilize two co-receptors for infection, but is similar to avian and murine oncoviruses, which use single receptors. This study sought to determine ELR1-binding domains of EIAV gp90. Towards this goal, a GFP-tagged gp90 fusion protein (gp90GFP) expression vector was constructed and a specific cell–cell binding assay was developed to measure EIAV gp90 binding to ELR1. Using these assays, the receptor-binding properties of 41 gp90GFP mutants were evaluated, each with a sequential replacement 11 aa linear epitope peptide from the vesicular stomatitis virus glycoprotein (VSV-G tag), as well as eight mutants containing individual gp90 variable-domain deletions. The results of these studies demonstrated that, in general, gp90 constructs containing substitutions or deletions in the N-terminal third of gp90 retained their receptor-binding activity. In contrast, segment substitutions or deletions in the C-terminal two-thirds of gp90 eliminated receptor-binding activity. Thus, these results reveal for the first time that the ELR1-binding domains of EIAV gp90 are located in the C-terminal two-thirds of EIAV gp90, apparently as a complex of discontinuous determinants.

Small-ruminant lentiviruses (SRLVs) display a high genetic diversity and are currently classified into five genotypes and an increasing number of subtypes. The co-circulation of subtypes in restricted geographical regions, combined with the occurrence of cross-species infection, suggests the need for development of a large-scale screening methodology for rapid monitoring of the prevalence of the various genetic subtypes and their genetic evolution. Here, a heteroduplex mobility assay (HMA) was developed for the rapid identification of group B subtypes. The assay was validated for both the p14 nucleocapsid-coding region of the gag gene and the V1–V2 region of the env gene using a panel of reference standards and was applied to the genetic subtyping of SRLV field isolates from five mixed flocks in France. Subtyping of 75 blood samples using the env HMA revealed a preferential distribution of subtypes B1 and B2 in sheep and goats, despite direct evidence for interspecies transmission of both subtypes. Adding the gag HMA to the env HMA provided evidence for dual infection and putative recombination between subtypes B1 and B2 in five goats, and between groups A and B in one sheep. Phylogenetic analysis revealed that 100 % (23/23) and 96.7 % (30/31) of samples were correctly classified using the gag and env HMAs, respectively. These results indicate that dual infection and recombination may be a significant source of new variation in SRLV and provide a useful tool for the rapid genetic subtyping of SRLV isolates, which could be relevant for the development of more accurate diagnosis of prevalent SRLV strains in different countries.

One of the major problems in gaining further insight into hepatitis B virus (HBV)/host-cell interactions is to improve the existing cellular models for the study of HBV replication. The first objective of this study was to improve the system based on transduction of HepG2 cells with a recombinant baculovirus to study HBV replication. A new HBV recombinant baculovirus, Bac-HBV-1.1, in which the synthesis of pre-genomic RNA is driven by a strong mammalian promoter, was generated. Transduction with this new recombinant baculovirus led to higher levels of HBV replication in HepG2 cells compared with levels obtained with previously described baculovirus vectors. The initiation of a complete HBV DNA replication cycle in Bac-HBV-1.1-transduced HepG2 cells was shown by the presence of HBV replicative intermediates, including covalently closed circular DNA (cccDNA). Only low levels of cccDNA were detected in the nucleus of infected cells. Data showed that cccDNA resulted from the recycling of newly synthesized nucleocapsids and was bound to acetylated histones in a chromatin-like structure. HBV particles released into the supernatant of transduced HepG2 cells were infectious in differentiated HepaRG cells. Several Bac-HBV-1.1 baculoviruses containing HBV strains carrying mutations conferring resistance to lamivudine and/or adefovir were constructed. Phenotypic analysis of these mutants confirmed the results obtained with the transfection procedures. In conclusion, an improved cell-culture system was established for the transduction of replication-competent HBV genomes. This will be useful for future studies of the fitness of HBV mutants.

Complete or almost complete hepatitis B virus (HBV) genomes were sequenced for 13 genotype A and 42 genotype D strains from the former USSR. The strains were classifiable within subgenotypes A2, D1, D2 and D3. Comparison of the deduced gene products for the four ORFs of 89 genotype D strains revealed 27 subgenotype-specific residues, and a region spanning residues 58–128 in the spacer region of the P gene could be used to distinguish between D1 and D4. This enabled the allocation to subgenotype of strains with partially sequenced genomes. D2 was dominating, while D3 was found in low frequency in the whole region. D1 was most prevalent in the Middle Asian Republics. Mean inter-subgenotype divergences between D1 and D2, D1 and D3 and D2 and D3 were 2.7, 3.4 and 3.4 %, respectively. The intra-subgenotype divergence was 0.4, 1.1, 1.0 and 1.8 % for A2, D1, D2 and D3, respectively. All D1 and D3 strains encoded subtype ayw2, whereas most D2 strains encoded ayw3. Two D2 strains encoded ayw4. Strains with identical S genes were closely related at the level of complete genomes and formed geographically specific clades with low intraclade divergences, possibly indicating past iatrogenic spread. It is not clear whether the finding of four subgenotypes in the area corresponds to separate introductions of the virus or to previous population migrations into the area. An earlier introduction of D3 compared with D2 was supported by its higher intra-subgenotype divergence, while the lower divergence within D1 is probably due to a more recent emergence.

Human parvovirus 4 (PARV4), a recently discovered parvovirus found exclusively in human plasma and liver tissue, was considered phylogenetically distinct from other parvoviruses. Here, we report the discovery of two novel parvoviruses closely related to PARV4, porcine hokovirus (PHoV) and bovine hokovirus (BHoV), from porcine and bovine samples in Hong Kong. Their nearly full-length sequences were also analysed. PARV4-like viruses were detected by PCR among 44.4 % (148/333) of porcine samples (including lymph nodes, liver, serum, nasopharyngeal and faecal samples), 13 % (4/32) of bovine spleen samples and 2 % (7/362) of human serum samples that were sent for human immunodeficiency virus and hepatitis C virus antibody tests. Three distinct parvoviruses were identified, including two novel parvoviruses, PHoV and BHoV, from porcine and bovine samples and PARV4 from humans, respectively. Analysis of genome sequences from seven PHoV strains, from three BHoV strains and from one PARV4 strain showed that the two animal parvoviruses were most similar to PARV4 with 61.5–63 % nt identities and, together with PARV4 (HHoV), formed a distinct cluster within the family Parvoviridae. The three parvoviruses also differed from other parvoviruses by their relatively large predicted VP1 protein and the presence of a small unique conserved putative protein. Based on these results, we propose a separate genus, Hokovirus, to describe these three parvoviruses. The co-detection of porcine reproductive and respiratory syndrome virus, the agent associated with the recent ‘high fever’ disease outbreaks in pigs in China, from our porcine samples warrants further investigation.