- Volume 84, Issue 10, 2003

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2·6–3·2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

Recombination is increasingly seen as an important means of shaping genetic diversity in RNA viruses. However, observed recombination frequencies vary widely among those viruses studied to date, with only sporadic occurrences reported in RNA viruses with negative-sense genomes. To determine the extent of homologous recombination in negative-sense RNA viruses, phylogenetic analyses of 79 gene sequence alignments from 35 negative-sense RNA viruses (a total of 2154 sequences) were carried out. Powerful evidence was found for recombination, in the form of incongruent phylogenetic trees between different gene regions, in only five sequences from Hantaan virus, Mumps virus and Newcastle disease virus. This is the first report of recombination in these viruses. More tentative evidence for recombination, where conflicting phylogenetic trees were observed (but were without strong bootstrap support) and/or where putative recombinant regions were very short, was found in three alignments from La Crosse virus and Puumala virus. Finally, patterns of sequence variation compatible with the action of recombination, but not definitive evidence for this process, were observed in a further ten viruses: Canine distemper virus, Crimean-Congo haemorrhagic fever virus, Influenza A virus, Influenza B virus, Influenza C virus, Lassa virus, Pirital virus, Rabies virus, Rift Valley Fever virus and Vesicular stomatitis virus. The possibility of recombination in these viruses should be investigated further. Overall, this study reveals that rates of homologous recombination in negative-sense RNA viruses are very much lower than those of mutation, with many viruses seemingly clonal on current data. Consequently, recombination rate is unlikely to be a trait that is set by natural selection to create advantageous or purge deleterious mutations.

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

Recombination plays a pivotal role in the evolutionary process of many different virus species, including retroviruses. Analysis of all human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants revealed that they are more complex than described initially. Recombination frequency is higher within certain genomic regions, such as partial reverse transcriptase (RT), vif/vpr, the first exons of tat/rev, vpu and gp41. A direct correlation was observed between recombination frequency and sequence similarity across the HIV-1 genome, indicating that sufficient sequence similarity is required upstream of the recombination breakpoint. This finding suggests that recombination in vivo may occur preferentially during reverse transcription through the strand displacement-assimilation model rather than the copy-choice model.

We present here a novel, distinct simian T-cell lymphotropic virus (STLV) found in a red-capped mangabey (Cercocebus torquatus) (CTO-NG409), wild-caught in Nigeria, that showed an HTLV-2-like Western blot (WB) seroreactivity. The complete genome (8920 bp) of CTO-NG409 STLV was related to but different from STLV-3/PHA-PH969 (13·5 %) and STLV-3/PPA-F3 (7·6 %), and STLV-3/CTO604 (11·3 %), found in Eritrean and Senegalese baboons, and red-capped mangabeys from Cameroon, respectively. Phylogenetic analysis of a conserved tax (180 bp) sequence and the env gene (1482 bp) confirmed the relatedness of STLV-3/CTO-NG409 to the STLV-3 subgroup. Molecular clock analysis of env estimated that STLV-3/CTO-NG409 diverged from East and West/Central African STLV-3s about 140 900±12 400 years ago, suggesting an ancient African origin of STLV-3. Since phylogenetic evidence suggests multiple interspecies transmissions of STLV-1 to humans, and given the antiquity and wide distribution of STLV-3 in Africa, a search for STLV-3 in human African populations with HTLV-2-like WB patterns is warranted.

Prevention of cross-species infection of porcine endogenous retroviruses (PERV) is crucial for xenotransplantation. The potential risk of infection is caused by replication-competent PERV as well as by hybrid viruses derived from recombination events of distinct PERV genomes. Recently, human-tropic, replication-competent PERV genomes obtaining hybrid sequences have been observed. Here, complete polymorphism pattern analysis was performed on the full-length PERV γ1 clones and on the complete envelope (env) gene sequences published to date. Several recombined full-length clones and a high number of different recombination patterns in the env gene were identified. In addition, recombinations with retroviral genomes not yet known were found. Thus, the potential risk of infection also exists for recombination products, including defective PERV loci.

To examine the mode of natural transmission and persistence of feline coronavirus (FCoV), FCoV strains shed by domestic cats were investigated over periods of up to 7 years. An RT-PCR that amplified part of the 3′ end of the viral spike (S) gene was devised to distinguish FCoV types I and II. All but 1 of 28 strains of FCoV from 43 cats were type I. Nucleotide identities of the amplified 320 bp product from 49 type I FCoVs ranged from 79 to 100 %. The consensus partial S sequence of isolates recovered from persistently infected cats at time intervals spanning years was generally conserved. While most cats were infected with a single strain, a few may have been infected by more than one strain. Cats that were transiently infected and ceased shedding could be re-infected with either the same, or a different, strain. In most cases, whether a cat became persistently or transiently infected was independent of the virus strain. However, one strain was unusual in that it infected the majority of cats in the household simultaneously and was still being shed 18 months later. Factors that influence whether FCoV establishes lifelong infection in some cats and not others are determined mainly by the host response to infection.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a 51Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain 51Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8+/CD4+ ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8+ T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus–host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

Recent studies suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system by upregulating interleukin (IL)-10 gene expression. To determine the effect of PRRSV on porcine cytokine gene expression in vivo, we infected pigs with either the European or North American strain of PRRSV and monitored cytokine gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) using a multiplex PCR assay. Our results showed that both European and North American strains of PRRSV significantly upregulated IL-10 gene expression in PBMC of infected pigs from 5 days post-infection (p.i.). In addition, upregulation of IL-10 and interferon (IFN)-γ gene expression was observed in BALC starting from 9 days p.i. The upregulation of cytokine gene expression in BALC was observed concurrent with an increased percentage of lymphocytes in the BALC population, suggesting a role for peripheral leukocytes in cytokine production in lungs. Our results showed that PRRSV infection resulted in an upregulation of IL-10 gene expression in vivo and that both European and North American strains induced comparable levels of IL-10 gene expression in the infected pigs, despite differences in the clinical signs. Our data support the notion that induction of IL-10 production may be one of the strategies used by PRRSV to modulate the host's immune responses, and this may contribute to the unique clinical picture observed following PRRSV infection.

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5′-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.

In this study, a serotype-specific monoclonal antibody (mAb), D2 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His–Arg/Lys–Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.

The induction of apoptotic cell death is a prominent cytopathic effect of dengue (DEN) viruses. One of the key questions to be addressed is which viral components induce apoptosis in DEN virus-infected cells. This study investigated whether the small membrane (M) protein was involved in the induction of apoptosis by DEN virus. This was addressed by using a series of enhanced green fluorescent protein-fused DEN proteins. Evidence is provided that intracellular production of the M ectodomains (residues M-1 to M-40) of all four DEN serotypes triggered apoptosis in host cells such as mouse neuroblastoma Neuro 2a and human hepatoma HepG2 cells. The M ectodomains of the wild-type strains of Japanese encephalitis, West Nile and yellow fever viruses also had proapoptotic properties. The export of the M ectodomain from the Golgi apparatus to the plasma membrane appeared to be essential for the initiation of apoptosis. The study found that anti-apoptosis protein Bcl-2 protected HepG2 cells against the death-promoting activity of the DEN M ectodomain. This suggests that the M ectodomain exerts its cytotoxic effects by activating a mitochondrial apoptotic pathway. The cytotoxicity of the DEN M ectodomain reflected the intrinsic proapoptotic properties of the nine carboxy-terminal amino acids (residues M-32 to M-40) designated ApoptoM. Residue M-36 was unique in that it modulated the death-promoting activity of the M ectodomain. Defining the ApoptoM-activated signalling pathways leading to apoptosis will provide the basis for studying how the M protein might play a key role in the fate of the flavivirus-infected cells.

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B–NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

The introduction and rapid dispersal of the African flavivirus West Nile virus (WNV) throughout North America, and the high fatality rate due to encephalitis in birds, horses, other wildlife species and humans, has attracted major attention worldwide. Usutu virus, another flavivirus, came to prominence in 2001, when it was identified as the agent responsible for a drop in the bird population in Austria; previously this encephalitic virus was found only in birds and mosquitoes in Africa. Sindbis virus, a pathogenic alphavirus that causes arthritis, is widespread throughout Africa, Europe, Asia and Australia, infecting a range of arthropods and vertebrates and is genetically related to encephalitic viruses in North America. Currently there is no evidence that any of these viruses cause disease in the UK. Here the presence of virus-specific neutralizing antibodies is reported in the sera of resident and migrant birds in the UK, implying that each of these viruses is being introduced to UK birds, possibly by mosquitoes. This is supported by nucleotide sequencing that identified three slightly different sequences of WNV RNA in tissues of magpies and a blackbird. The detection of specific neutralizing antibodies to WNV in birds provides a plausible explanation for the lack of evidence of a decrease in the bird population in the UK compared with North America. The potential health risk posed to humans and animals by these viruses circulating in the UK is discussed.

The pathogenesis of poliovirus infection, responsible for the induction of a poliovirus-specific mucosal immune response following intraperitoneal (i.p.) inoculation of virus in mice transgenic for the poliovirus receptor (PVRTg mice), was studied. Following inoculation of poliovirus, replication was determined by increase in virus titre (TCID50) and by PCR of poliovirus-specific negative-strand RNA in peritoneal macrophages, mesenteric lymph nodes, Peyer's patches, duodenum, brain, kidney and liver. The presence of poliovirus antigens in several cell types was detected by immunolabelling. It was demonstrated that poliovirus replicated in the peritoneal macrophages of PVRTg mice, since the virus titre in peritoneal cells was increased compared to the titre in the inoculum. Negative-strand RNA was detected in these cells and most of the poliovirus-immunostained cells had the morphology of macrophages and expressed the macrophage-specific markers CD86 and M1/70 on their surface. Furthermore, in peritoneal lavage, poliovirus was also present in CD19+ B cells, but not in dendritic or T cells. Moreover, poliovirus was detected in macrophage-like cells in the lamina propria of the intestine, but not in epithelial cells. Replication of poliovirus in mesenteric lymph nodes, Peyer's patches and brain was followed by excretion of virus in the faeces. This suggests that the virus is transported due to migration of macrophages from the peritoneal cavity to mesenteric lymph nodes and the lamina propria of Peyer's patches. It is likely that this route is responsible for the induction of virus-specific IgA in the gut.

Human rhinoviruses (HRV) are responsible for the majority of virus infections of the upper respiratory tract. Furthermore, HRV infection is associated with acute exacerbation of asthma and other chronic respiratory diseases of the lower respiratory tract. A small animal model of HRV-induced disease is required for the development of new therapies. However, existing mouse models of HRV infection are difficult to work with and until recently mouse cell lines were thought to be generally non-permissive for HRV replication in vitro. In this report we demonstrate that a virus of the minor receptor group, HRV1B, can infect and replicate in a mouse respiratory epithelial cell line (LA-4) more efficiently than in a mouse fibroblast cell line (L). The major receptor group virus HRV16 requires human intercellular adhesion molecule-1 (ICAM-1) for cell entry and therefore cannot infect LA-4 cells. However, transfection of in vitro-transcribed HRV16 RNA resulted in the replication of viral RNA and production of infectious virus. Expression of a chimeric ICAM-1 molecule, comprising mouse ICAM-1 with extracellular domains 1 and 2 replaced by the equivalent human domains, rendered the otherwise non-permissive mouse respiratory epithelial cell line susceptible to entry and efficient replication of HRV16. These observations suggest that the development of mouse models of respiratory tract infection by major as well as minor group HRV should be pursued.

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal ∼30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria mellonella larvae.

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that spreads in the nervous system in functionally connected neurons. Determining how HSV-1 components are sorted in neurons is critical to elucidate the mechanisms of virus neuroinvasion. By using recombinant viruses expressing glycoprotein B (gB) tagged with green fluorescent protein (GFP), the subcellular localization of this envelope protein was visualized in infected hippocampal neurons in culture. Results obtained using a fully infectious recombinant virus containing GFP inserted into the ectodomain of gB support the view that capsids and gB are transported separately in neuron processes. Moreover, they show that during infection gB is sorted to the dendritic tree and the axons of polarized hippocampal neurons. However, GFP insertion into the cytoplasmic tail of gB impaired the maturation of the resulting fusion protein and caused its retention in the endoplasmic reticulum. The defective protein did not gain access to axons of infected neurons. These results suggest that the cytoplasmic tail of gB plays a role in maturation and transport and subsequently in axonal sorting in differentiated hippocampal neurons.