- Volume 89, Issue 11, 2008

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.

The unprecedented emergence in Asia of multiple avian influenza virus (AIV) subtypes with a broad host range poses a major challenge in the design of vaccination strategies that are both effective and available in a timely manner. The present study focused on the protective effects of a genetically modified AIV as a source for the preparation of vaccines for epidemic and pandemic influenza. It has previously been demonstrated that a live attenuated AIV based on the internal backbone of influenza A/Guinea fowl/Hong Kong/WF10/99 (H9N2), called WF10att, is effective at protecting poultry species against low- and high-pathogenicity influenza strains. More importantly, this live attenuated virus provided effective protection when administered in ovo. In order to characterize the WF10att backbone further for use in epidemic and pandemic influenza vaccines, this study evaluated its protective effects in mice. Intranasal inoculation of modified attenuated viruses in mice provided adequate protective immunity against homologous lethal challenges with both the wild-type influenza A/WSN/33 (H1N1) and A/Vietnam/1203/04 (H5N1) viruses. Adequate heterotypic immunity was also observed in mice vaccinated with modified attenuated viruses carrying H7N2 surface proteins. The results presented in this report suggest that the internal genes of a genetically modified AIV confer similar protection in a mouse model and thus could be used as a master donor strain for the generation of live attenuated vaccines for epidemic and pandemic influenza.

Since early 2007, the Kingdom of Saudi Arabia (KSA) has experienced several highly pathogenic avian influenza (HPAI) H5N1 outbreaks in the falconry and poultry sectors. The public health threat associated with peculiar husbandry systems, requiring close contact between humans and birds of prey, highlights the need of an improved understanding of the epidemiology and of the viral characteristics of H5N1 viruses circulating in the region. Here we report molecular and phylogenetic analyses of H5N1 viruses isolated in the KSA in 2007 in distinct compartments of avian husbandry. From the results of our investigation it appears that two separate introductions into the different sectors occurred. The identification of specific amino acid mutations, which are described as genetic signatures of human influenza A viruses or known to confer resistance to antiviral drugs, raises concerns for the possible human health implications of the KSA H5N1 viruses.

Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.

BALB/c mice infected with human metapneumovirus (hMPV) were treated with the sulfated sialyl lipid NMSO3 (one dose of 50 mg kg−1) given at the time of infection. NMSO3 significantly reduced viral replication in the lungs, as well as hMPV-induced body weight loss, pulmonary inflammation and cytokine production, suggesting that antiviral treatment initiated at the beginning of viral infection can modify hMPV-induced disease.

α-Dystroglycan (α-DG) is a ubiquitously expressed molecule that has been identified as a cellular receptor for lymphocytic choriomeningitis virus (LCMV) and other arenaviruses. Recently, it was demonstrated that LCMV receptor function is critically dependent on post-translational modifications, namely glycosylation. In particular, it was shown that O-mannosylation, a rare type of mammalian O-linked glycosylation, is important in determining the binding of LCMV to its cellular receptor. All studies carried out so far showed a dependence on glycosylation in LCMV receptor function in vitro. This work extended these studies to two in vivo models of α-DG hypoglycosylation. The results confirm earlier findings on the in vitro dependence of carbohydrate modifications in LCMV receptor function. However, experiments in animal models showed that this dependence was only very weak in vivo. It is likely that alternative receptors or alternative entry pathways may account for this attenuated in vivo phenotype.

Human hantaviral disease is mediated by excessive proinflammatory and CD8+ T-cell responses, which can be alleviated by administration of corticosteroids. In contrast with humans, male rats that are infected with their species-specific hantavirus, Seoul virus (SEOV), have reduced proinflammatory and elevated regulatory T-cell responses in tissues where virus persists. To determine the effects of glucocorticoids on SEOV persistence and immune responses during infection, male and female Norway rats received sham surgeries (sham) or were adrenalectomized (ADX0), in some of which corticosterone was replaced at low (ADX10) or high (ADX80) doses. Rats were inoculated with SEOV and serum corticosterone, SEOV RNA, gene expression and protein production were measured at different time points post-inoculation. We observed that SEOV infection suppressed corticosterone in sham males to concentrations seen in ADX0 males. Furthermore, males with low corticosterone had more SEOV RNA in the lungs than either females or males with high corticosterone concentrations during peak infection. Although high concentrations of corticosterone suppressed the expression of innate antiviral and proinflammatory mediators to a greater extent in females than in males, these immunomodulatory effects did not correlate with SEOV load. Males with low corticosterone concentrations and high viral load had elevated regulatory T-cell responses and expression of matrix metalloprotease (MMP)-9. MMP-9 is a glycogenase that disrupts cellular matrices and may facilitate extravasation of SEOV-infected cells from circulation into lung tissue. Suppression of glucocorticoids may thus contribute to more efficient dissemination of SEOV in male than in female rats.

Infection with feline infectious peritonitis virus (FIPV), a feline coronavirus, frequently leads to death in spite of a strong humoral immune response. In previous work, we reported that infected monocytes, the in vivo target cells of FIPV, express viral proteins in their plasma membranes. These proteins are quickly internalized upon binding of antibodies. As the cell surface is cleared from viral proteins, internalization might offer protection against antibody-dependent cell lysis. Here, the internalization and subsequent trafficking of the antigen–antibody complexes were characterized using biochemical, cell biological and genetic approaches. Internalization occurred through a clathrin- and caveolae-independent pathway that did not require dynamin, rafts, actin or rho-GTPases. These findings indicate that the viral antigen–antibody complexes were not internalized through any of the previously described pathways. Further characterization showed that this internalization process was independent from phosphatases and tyrosine kinases but did depend on serine/threonine kinases. After internalization, the viral antigen–antibody complexes passed through the early endosomes, where they resided only briefly, and accumulated in the late endosomes. Between 30 and 60 min after antibody addition, the complexes left the late endosomes but were not degraded in the lysosomes. This study reveals what is probably a new internalization pathway into primary monocytes, confirming once more the complexity of endocytic processes.

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

The biochemical events triggered by viral infection are critical to the outcome of a host immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) causes the most significant disease of swine worldwide. Onset of infection is insidious and subclinical. Clinical signs may not appear for days and antibodies cannot be detected for a week or more. To understand better the early pathophysiological response of swine to PRRSV infection and its inapparent onset, we examined serum samples in the first days of infection for evidence of early biochemical changes. Sera from pigs infected with various isolates of PRRSV were extracted to remove high molecular mass proteins, desalted and analysed by matrix assisted laser desorption/ionization–time of flight mass spectrometry (MS). Comparative analysis of low molecular mass serum protein profiles revealed that one protein, with an m/z value of 9244±2, appeared within 1 day of infection. The 9244±2 peak was identified as the alpha 1S (α1S)-subunit of porcine haptoglobin (Hp) by tandem MS sequencing and confirmed by immunoblotting with anti-porcine Hp antibody. Hp is an acute phase haem-binding protein consisting of α–β heterodimers that is secreted from the liver in response to stresses, including infection. However, the presence of free α1S-subunit in response to infection is novel and may provide new insights into biochemical processing of Hp and its role in disease pathogenesis, including PRRS.

The largest documented outbreak of Chikungunya virus (CHIKV) disease occurred in the Indian Ocean islands and India during 2004–2007. The magnitude of this outbreak led to speculation that a new variant of the virus had emerged that was either more virulent or more easily transmitted by mosquito vectors. To study this assertion, it is important to know the origin of the virus and how the particular strain circulating during the outbreak is related to other known strains. This study genetically characterized isolates of CHIKV obtained from Mombasa and Lamu Island, Kenya, during 2004, as well as strains from the 2005 outbreak recorded in Comoros. The results of these analyses demonstrated that the virus responsible for the epidemic that spread through the Indian Ocean originated in coastal Kenya during 2004 and that the closest known ancestors are members of the Central/East African clade. Genetic elements that may be responsible for the scope of the outbreak were also identified.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Viral infection of mammalian cells prompts the innate immune system to initiate an antiviral response. The recognition of the virus triggers several antiviral signalling pathways, which among others include the family of 2′-5′ oligoadenylate synthetase (OAS) proteins. The p59 protein encoded by the OAS-like (OASL) gene is an atypical member of the OAS family in the sense that it lacks the characteristic 2′-5′ oligoadenylate synthetase activity. We decided to investigate the putative antiviral activity of p59 by ectopically expressing this protein in Vero cells and then infecting these cells with virus. We demonstrate that OASL has an antiviral effect against the single-stranded RNA virus picornavirus, encephalomyocarditis virus, but not against a large DNA virus, herpes simplex virus 1. Importantly, this antiviral activity was lost in a truncated version of p59 lacking the ubiquitin-like C-terminal domain of p59. Taken together our results indicate that p59 is indeed an antiviral protein that works through a novel mechanism distinct from other OAS proteins.

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) as described for women with an established infection is, in most cases, associated with the transmission of few maternal variants. This study analysed virus variability in four cases of maternal primary infection occurring during pregnancy and/or breastfeeding. Estimated time of seroconversion was at 4 months of pregnancy for one woman (early seroconversion) and during the last months of pregnancy and/or breastfeeding for the remaining three (late seroconversion). The C2V3 envelope region was analysed in samples of mother–child pairs by molecular cloning and sequencing. Comparisons of nucleotide and amino acid sequences as well as phylogenetic analysis were performed. The results showed low variability in the virus population of both mother and child. Maximum-likelihood analysis showed that, in the early pregnancy seroconversion case, a minor viral variant with further evolution in the child was transmitted, which could indicate a selection event in MTCT or a stochastic event, whereas in the late seroconversion cases, the mother's and child's sequences were intermingled, which is compatible with the transmission of multiple viral variants from the mother's major population. These results could be explained by the less pronounced selective pressure exerted by the immune system in the early stages of the mother's infection, which could play a role in MTCT of HIV-1.

Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein transforms human lymphocytes and is critical for the pathogenesis of HTLV-1-induced adult T-cell leukaemia. In HTLV-transformed cells, Tax upregulates interleukin (IL)-13, a cytokine with proliferative and anti-apoptotic functions that is linked to leukaemogenesis. Tax-stimulated IL-13 is thought to result in autocrine stimulation of HTLV-infected cells and thus may be relevant to their growth. The causal transactivation of the IL-13 promoter by Tax is predominantly dependent on a nuclear factor of activated T cells (NFAT)-binding P element. Here, it was shown that the isolated IL-13 Tax-responsive element (IL13TaxRE) was sufficient to mediate IL-13 transactivation by Tax and NFAT1. However, cyclosporin A, a specific NFAT inhibitor, revealed that Tax transactivation of IL13TaxRE or wild-type IL-13 promoter was independent of NFAT and that NFAT did not contribute to IL-13 upregulation in HTLV-transformed cells. By contrast, Tax stimulation was repressible by an efficient nuclear factor (NF)-κB inhibitor (IkBaDN), indicating the requirement for NF-κB. The capacity of NF-κB to stimulate IL13TaxRE was demonstrated by a strong response to NF-κB in reporter assays and by direct binding of NF-κB to IL13TaxRE. Thus, IL13TaxRE in the IL-13 promoter represents a dually active promoter element responsive to NF-κB and NFAT. Together, these results indicate that Tax causes IL-13 upregulation in HTLV-1-infected cells via NF-κB.

A nested-PCR (n-PCR) was used to detect feline leukemia virus (FeLV) proviral DNA in blood samples from 464 sick and 608 healthy domestic cats (Felis catus) selected by convenience, and a significantly high prevalence of FeLV infection was observed. n-PCR results revealed the presence of FeLV proviral DNA in 47.2 % of sick cats and 47.4 % of healthy cats. Phylogenetic analysis revealed that FeLV samples from healthy or sick cats were grouped into separate clades. We determined FeLV subgroups by an n-PCR based on the envelope (env) gene. The partial env gene of FeLV Minas Gerais (MG) samples were compared to various exogenous FeLV isolates and endogenous (enFeLV) provirus from the same region. FeLV-B MG samples were more similar to endogenous sequences and to natural FeLV-B isolates than to either FeLV-A or FeLV-C. The results revealed the circulation of FeLV-B in large populations of urban domestic cats in Brazil.

SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein–Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell–cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1β and IL-1α was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.

Epstein–Barr virus nuclear antigen-1 (EBNA-1) is the only latent protein expressed in all virus-associated tumours. It plays a critical role in viral propagation and in the replication, episomal maintenance and partitioning of the viral genome. However, its tumorigenic potential is debated. We have previously shown that lymphocytes from a tumour-prone, EBNA-1-expressing, transgenic mouse line show increased responsiveness to interleukin-2 (IL-2). It was important to determine whether this property was unique to the transgenic line or whether it is a general consequence of EBNA-1 expression in B cells. In order to distinguish between these possibilities, explanted lymphocytes from two independent transgenic mouse lines were examined. The lymphocytes from both lines showed enhanced proliferation rates compared with controls. The transgenic lymphocytes survived for extended periods in culture, dependent on the dose of IL-2, while IL-15 (the receptor of which shares the β and γ chain components of the IL-2 receptor) induced little effect. In accordance with this, transgenic B cells showed enhanced induction of expression of the IL-2 receptor α chain (CD25), which modulates affinity for the ligand. As this phenotype is evident in lymphocytes from mice of both lines, it is necessarily independent of any transgene insertion site effects and may be attributed to EBNA-1 expression. Furthermore, 10/12 tumour-bearing transgenic mice had elevated IL-2 levels in serum and 4/6 tumours were CD25 positive. IL-2 is normally produced by activated T cells in vivo; thus, chronic immune activation or modulation could elicit this unique mode of virus-infected cell survival.