- Volume 87, Issue 8, 2006

Hepatitis C virus (HCV) interacts with human platelets in vivo as a potential transport of infectious virions to the target liver. The binding of native viral particles with the platelet membrane glycoprotein VI (GPVI) was analysed. A consistent interaction between HCV from plasma or after purification by two different methods and the recombinant extracellular immunoglobulin (Ig)-like domains of human GPVI (hD1D2) was observed with two independent experimental approaches: pull-down and ELISA assays. Between 2 and 7 % of HCV particles were specifically bound to hD1D2. The binding was inhibited by an anti-hD1D2 in a dose-dependent manner. Human D1D2 interaction with HCV was significantly higher than the murine D1D2, supporting the specificity of the interaction and to the single human domains (D1 and D2), suggesting that both Ig-like domains of the molecule are required for efficient binding. GPVI may be a platelet surface ligand for HCV playing a role in viral transport and persistence.

Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-γ responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-γ responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.

A full-length infectious cDNA clone (ic) was constructed from the genome of the dengue virus type 2 (DENV-2) Jamaica83 1409 strain, pBAC1409ic, by using a bacterial artifical chromosome plasmid system. Infectious virus was generated and characterized for growth in cell culture and for infection in Aedes aegypti mosquitoes. During construction, an isoleucine to methionine (Ile→Met) change was found at position 6 in the envelope glycoprotein sequence between low- and high-passage DENV-2 1409 strains. In vitro-transcribed genomic RNA of 1409ic with E6-Ile produced infectious virions following electroporation in mosquito cells, but not mammalian cells, while 1409ic RNA with an E6-Met mutation produced virus in both cell types. Moreover, DENV-2 1409 with the E6-Ile residue produced syncytia in C6/36 cell culture, whereas viruses with E6-Met did not. However, in vitro cell culture-derived growth-curve data and in vivo mosquito-infection rates revealed that none of the analysed DENV-2 strains differed from each other.

The human placenta is relatively resistant to Human immunodeficiency virus 1 (HIV-1), but obstetric complications associated with inflammatory processes, including chorioamnionitis and spontaneous preterm delivery, are associated with increased rates of vertical transmission. It was hypothesized that the pro-inflammatory mediator tumour necrosis factor alpha (TNF-α), which promotes HIV-1 transmission across endothelial membranes, increases HIV-1 transmission across the placenta. Flow cytometry and immunostaining studies were performed, which demonstrated that the HIV-1 receptors CD4, CCR5 and CXCR4 were not expressed by villous trophoblast cells. Consequently, primary villous trophoblast cells were not infected with cell-free HIV-1 isolates, as measured by in situ PCR and quantitative PCR, but villous trophoblast cells were infected by HIV-1-infected peripheral blood mononuclear cells (PBMC). HIV-1 from infected PBMC was rapidly transported across confluent transformed trophoblast cell monolayers by transcytosis, and TNF-α significantly upregulated transcytosis of HIV-1 across the trophoblast layer without disrupting cell viability or confluency. Inhibitors of TNF-α (antibodies against TNF-α and TNF-α receptors) and an anti-inflammatory drug (tenidap) significantly reduced transcytosis rates. It was concluded that the villous trophoblast is resistant to infection by cell-free HIV-1 but susceptible to transcytosis of HIV-1 from infected PBMC, and inflammatory mediators such as TNF-α may play a critical role in promoting maternal–fetal transmission of HIV-1.

A retroviral vector-rescue system in which co-packaging of the two co-expressed vectors is required for transduction of one of the vectors has been established previously. By using this rescue system, two distinct packaging-cell populations have been generated. One cell population expressed retroviral RNA from co-localized transcription sites, resulting in local and overlapping accumulation of both RNA transcripts. In the other cell population, the two transcription cassettes were introduced separately, leading to distinct transcription sites of the two RNAs and no significant co-localization of the RNAs. Titre measurements from the two distinct cell populations showed large differences in rescue titre, which is an indirect measure of co-packaging efficiency. Thus, the cell populations with overlapping RNA accumulation gave rise to 15–80-fold-higher rescue titres than cell populations with non-overlapping RNA accumulation. These data show that the spatial position of proviral transcription sites affects the level of retroviral RNA co-packaging and suggest that there is already a linkage of RNAs for co-packaging at the transcription site. It is hypothesized that this linkage is due to RNA dimerization taking place at the transcription site.

The Nef protein is a crucial pathogenicity factor of human immunodeficiency virus type 1 (HIV-1) that contains a proline-rich motif consisting of four conserved prolines: Pro69 (P69), P72, P75 and P78. P72 and P75 were shown to bind Src homology domains 3 (SH3) and have been implicated in many biological functions of Nef, including downmodulation of cell-surface major histocompatibility complex class I (MHC-I). P78 is involved together with P69 in positioning of the Nef–SH3 complex and it has been shown to be essential for Nef activity of MHC-I downmodulation. It is shown here that alteration of P78 affects recycling of MHC-I molecules to the cell surface, but does not interfere with SH3 binding. In addition, it is demonstrated that P72 and P75, and thus the SH3-binding capacity, are fully dispensable for Nef activity on MHC-I.

Substitution of lentiviral cis-acting elements by heterologous sequences might allow the safety of lentiviral vectors to be enhanced by reducing the risk of homologous recombination and vector mobilization. Therefore, a substitution and deletion analysis of the R region of simian immunodeficiency virus (SIV)-based vectors was performed and the effect of the modifications on packaging and transfer by SIV and human immunodeficiency virus type 1 (HIV-1) particles was analysed. Deletion of the first 7 nt of R reduced vector titres by 10- to 20-fold, whilst deletion of the entire R region led to vector titres that were 1500-fold lower. Replacement of the R region of SIV-based vectors by HIV-1 or Moloney murine sarcoma virus R regions partially restored vector titres. A non-retroviral cellular sequence was also functional, although to a lesser extent. In the absence of tat, modification of the R region had only minor effects on cytoplasmic RNA stability, steady-state levels of vector RNA and packaging, consistent with the known primary function of R during reverse transcription. Although the SIV R region of SIV-based vectors could be replaced functionally by heterologous sequences, the same modifications of R led to a severe replication defect in the context of a replication-competent SIV. As SIV-based vectors containing the HIV-1 R region were transferred less efficiently by HIV-1 particles than wild-type SIV vectors, a match between R and cis-acting elements of the vector construct seems to be more important than a match between R and the Gag or Pol proteins of the vector particle.

Bats represent the major source of human rabies cases in the New World. In the USA, most cases are associated with species that are not commonly found or reported rabid. To understand better the epidemiology and public health significance of potentially important bat species, a molecular study was performed on samples collected from naturally infected rabid western pipistrelle (Pipistrellus hesperus), eastern pipistrelle (Pipistrellus subflavus) and silver-haired bats (Lasionycteris noctivagans) from different regions of their geographical distribution in the USA. A 264 bp fragment at the 5′ end of the N gene coding region was sequenced and analysed in comparison with rabies virus variants circulating within other North American mammals. Phylogenetic analysis demonstrated that P. hesperus bats maintain a unique rabies virus variant. Preliminary data also suggest that P. subflavus and Lasionycteris noctivagans may harbour two different rabies virus variants (Ps and Ln) that are likely to be maintained independently by each bat species, which recently appear to have emerged as major vectors of human disease.

Partial nucleoprotein (N) gene sequences of the rhabdoviruses Obodhiang (OBOV), Kotonkon (KOTV), Rochambeau (RBUV), Kern canyon (KCV), Mount Elgon bat (MEBV), Kolongo (KOLV) and Sandjimba (SJAV) were generated and their phylogenetic positions within the family Rhabdoviridae were determined. Both OBOV and KOTV were placed within the genus Ephemerovirus. RBUV was joined to the same cluster, but more distantly. MEBV and KCV were grouped into a monophyletic cluster (putative genus) with Oita virus (OITAV). These three viruses, originating from different regions of the world, were all isolated from insectivorous bats and may be specific for these mammals. African avian viruses KOLV and SJAV were joined to each other and formed another clade at the genus level. Further, they were grouped with the recently characterized rhabdovirus Tupaia virus (TRV). Although the genetic distance was great, the grouping was supported by consistent bootstrap values. This observation suggests that viruses of this group may be distributed widely in the Old World. Non-synonymous/synonymous substitution ratio estimations (d N/d S) using a partial N gene fragment (241 codons) for the three rhabdovirus genera revealed contrasting patterns of evolution, where d N/d S values follow the pattern Ephemerovirus > Vesiculovirus > Lyssavirus. The magnitude of this ratio corresponds well with the number of negatively selected codons. The accumulation of d S appears evenly distributed along the gene fragment for all three genera. These estimations demonstrated clearly that lyssaviruses are subjected to the strongest constraints against amino acid substitutions, probably related to their particular niche and unique pathobiology.

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus–host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus–model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.

Porcine reproductive and respiratory syndrome virus (PRRSV) can evade the host immune system, which results in prolonged virus replication for several weeks to several months. To date, the mechanisms of PRRSV immune evasion have not been investigated in detail. One possible immune-evasion strategy is to avoid incorporation of viral proteins into the plasma membrane of infected cells, as this prevents recognition by virus-specific antibodies and consequent cell lysis either by the classical complement pathway or by antibody-dependent, cell-mediated cytotoxicity. In this study, viral proteins were not observed in the plasma membrane of in vitro-infected macrophages by using confocal microscopy or flow cytometry. Subsequently, the sensitivity of PRRSV-infected macrophages towards antibody-dependent, complement-mediated cell lysis (ADCML) was determined by using an ADCML assay. A non-significant percentage of PRRSV-infected cells were killed in the assay, showing that in vitro PRRSV-infected macrophages are protected against ADCML. PRRSV proteins were not detected in the plasma membrane of in vivo-infected alveolar macrophages and ADCML was also not observed. Together, these data indicate that viral proteins are not incorporated into the plasma membrane of PRRSV-infected macrophages, which makes infected cells invisible to PRRSV-specific antibodies. This absence of viral proteins on the cell surface could explain the protection against ADCML observed for in vitro and in vivo PRRSV-infected macrophages, and may play a role in virus persistence.

Variation in infectivity and genetic diversity in the structural proteins were compared among eight strains of Western equine encephalitis virus (WEEV) to investigate WEEV virulence at the molecular level. A lethal intranasal infectivity model of WEEV was developed in adult BALB/c mice. All eight strains examined were 100 % lethal to adult mice in this model, but they varied considerably in the time to death. Based on the time to death, the eight strains could be classified into two pathotypes: a high-virulence pathotype, consisting of strains California, Fleming and McMillan, and a low-virulence pathotype, comprising strains CBA87, Mn548, B11, Mn520 and 71V-1658. To analyse genetic diversity in the structural protein genes, 26S RNAs from these eight strains were cloned and sequenced and found to have >96 % nucleotide and amino acid identity. A cluster diagram divided the eight WEEV strains into two genotypes that matched the pathotype grouping exactly, suggesting that variation in infectivity can be attributed to genetic diversity in the structural proteins among these eight strains. Furthermore, potential amino acid differences in some positions between the two groups were identified, suggesting that these amino acid variations contributed to the observed differences in virulence.

Full-length sequences were determined for a human hepatitis E virus (HEV) isolate (HE-JA04-1911) and two swine HEV isolates (swJ8-5 and swJ12-4) that belong to one of three clusters within genotype 3 in Japan and are close to Spanish isolates according to their partial sequences. The three HEV isolates were 89.7–92.9 % identical to each other, but only 80.7–83.0 % similar to 21 HEV strains of the same genotype isolated in Canada, Kyrgyzstan, the USA and Japan over their entire genome. On comparison with HEV isolates whose partial sequence is known, the HE-JA04-1911, swJ8-5 and swJ12-4 isolates segregated into a phylogenetic cluster consisting of human and swine HEV isolates in Japan and the UK, with identities of 89.8–100 % and 87.9–92.4 %, respectively. Genotype 3 HEV isolates were found to be markedly heterogeneous. The UK-isolate-like HEV strains in Japan may have originated from the UK via the importation of pigs since 1900.

Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained at high frequency in experimentally coinfected cattle, the consequences of recombination on the virulence of gE-negative BoHV-1 were investigated. Thus, gE-negative BoHV-1 recombinants were generated in vitro from several virulent BoHV-1 and one mutant BoHV-1 deleted in the gC and gE genes. Four gE-negative recombinants were tested in the natural host. All the recombinants were more virulent than the gE-negative BoHV-1 vaccine and the gC- and gE-negative parental BoHV-1. The gE-negative recombinant isolated from a BoHV-1 field strain induced the highest severe clinical score. Latency and reactivation studies showed that three of the recombinants were reexcreted. Recombination can therefore restore virulence of gE-negative BoHV-1 by introducing the gE deletion into a different virulence background.

Previous studies have identified virus proteins that traffic to mitochondria and may affect mitochondrial function. Here, it is reported that Human herpesvirus 1 (HHV-1, herpes simplex virus 1) and influenza virus reduced mitochondrial respiration, whilst Measles virus, cytomegalovirus, coxsackievirus B4 and Feline calicivirus did not. The inhibition of total cellular respiration was caused by a block in the mitochondrial electron-transport chain. This effect occurred during β-phase protein synthesis and the inhibition of mitochondrial respiration could be reproduced by ectopic expression of the β-phase protein US3. An HHV-1 mutant lacking this protein failed to inhibit oxygen consumption in infected cells relative to controls. It was concluded that US3 was mediating the suppression of mitochondrial respiration following HHV-1 infection. The integrity of the electron-transport chain in HHV-1-infected cells was analysed further and the site of the block in electron transport was located between complexes II and III, a site previously shown to be affected by Poliovirus.

Toll-like receptor 3 (TLR-3) and TLR-9 gene expression and interleukin 6 (IL-6) secretion were studied in corneal cells with components of herpes simplex virus (HSV). Human corneal epithelial cells (HCEs) and primary human corneal fibroblasts (HCRFs) were infected with live HSV or UV-inactivated HSV (UV-HSV), transfected with HSV DNA or treated with HSV–anti-HSV IgG immune complexes. Gene expression of TLR-3 and -9 was analysed by real-time PCR. Supernatants were assayed for IL-6 by ELISA. Incubation of HCEs and HCRFs with live HSV-1, UV-HSV and HSV DNA resulted in augmented TLR-3 and -9 gene expression and IL-6 release. Moreover, infected or transfected HCRFs released greater amounts of IL-6 than did HCEs. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. Here, it is shown that HSV–anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. Treatment of HCRFs transfected with HSV DNA with the TLR-9-inhibitory oligomer iODN, anti-TLR-3 antibody or phosphatidylinositol 3-kinase inhibitor indicated that IL-6 release from HCRFs was mediated by TLR-3 and -9 gene expression. These results demonstrated that neutralized HSV immune complexes were as potent as HSV DNA in enhancing IL-6 release from corneal fibroblasts. These phenomena were mediated via augmented TLR-3 and -9 gene expression.

For some time there has been evidence suggesting an interaction between human cytomegalovirus (HCMV) and Human immunodeficiency virus (HIV) in the pathogenesis of AIDS. Here, the interaction of HCMV and HIV-1 was examined in monocyte/macrophage cells, two cell types known to be targets for both viruses in vivo. Infection experiments demonstrated that prior infection with HCMV impeded subsequent superinfection with HIV-1. In contrast, uninfected bystander cells within the population were still permissive for HIV-1 infection and were also found to express increased levels of Gag after HIV-1 superinfection. Analysis of CCR5, a co-receptor for HIV-1, on HCMV-infected and bystander cells showed a substantial loss of surface CCR5 expression on infected cells due to HCMV-induced reduction of total cellular CCR5. In contrast, uninfected bystander cells displayed increased surface CCR5 expression. Furthermore, the data suggested that soluble factor(s) secreted from HCMV-infected cells were responsible for the observed upregulation of CCR5 on uninfected bystander cells. Taken together, these results suggest that, whilst HCMV-infected monocytes/macrophages are refractory to infection with HIV-1, HCMV-uninfected bystander cells within a population are more susceptible to HIV-1 infection. On this basis, HCMV infection may contribute to the pathogenesis of HIV-1.

In human cytomegalovirus-infected cells, the immediate-early IE1 protein disrupts the subnuclear structures known as the PML oncogenic domains or PODs, via the induction of PML desumoylation. This activity correlates with the functions of IE1 in transcriptional regulation and in the stimulation of lytic infection. Here, the effects of IE1 in induction of desumoylation of PML were characterized. IE1 did not interfere with the formation of sumoylated forms of PML in vitro. In in vitro assays using the sumoylated proteins, a SUMO-specific protease SENP1 desumoylated both PML and IE1. However, the IE1 proteins generated from bacteria or insect cells were unable to desumoylate PML in the same conditions. Although both IE1 and SUMO proteases such as SENP1, Axam and SuPr-1 efficiently desumoylated PML in co-transfection assays, they exerted different effects on the localization of PML. In cells transfected with either SENP1 or SuPr-1, the number of PML foci was reduced significantly and these remnant PML foci were devoid of SUMO-1 signals. However, in cells co-transfected with both SUMO proteases and IE1, these SUMO-independent PML foci were also completely disrupted. Furthermore, IE1, but not SENP1, was shown to disrupt the PML foci generated via transfection of a sumoylation-deficient mutant of PML. These data suggest that IE1 exhibits neither an inhibitory effect on sumoylation of PML nor intrinsic SUMO protease activity against PML in vitro. The finding that IE1 is capable of disrupting SUMO-independent PML aggregates suggests that inhibition of PML oligomerization by IE1 may play an important role in inducing PML desumoylation in vivo.

Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.