- Volume 88, Issue 12, 2007

Between 2000 and 2004, dengue virus type 1 (DENV-1) genotypes I and II from Asia were introduced into the Pacific region and co-circulated in some localities. Envelope protein gene sequences of DENV-1 from 12 patients infected on the island of New Caledonia were obtained, five of which carried genotype I viruses and six, genotype II viruses. One patient harboured a mixed infection, containing viruses assigned to both genotypes I and II, as well as a number of inter-genotypic recombinants. This is the first report of a population of dengue viruses isolated from a patient containing both parental and recombinant viruses.

GB virus type C (GBV-C) is a common human flavivirus that has been associated with prolonged survival in HIV-positive individuals in several, though not all, epidemiological studies. There are five distinct GBV-C genotypes that are geographically localized, and it has been speculated that GBV-C genotypic differences may explain variable outcomes observed in different clinical studies. Expression of an 85 aa fragment of the GBV-C NS5A phosphoprotein (genotype 2) in a CD4+ T cell line (Jurkat) resulted in inhibition of HIV replication, mediated in part by decreased surface expression of the HIV coreceptor CXCR4 and upregulation of SDF-1. We expressed the NS5A protein from genotypes 1, 2, 3 and 5 in Jurkat cells, and demonstrated that all genotypes inhibited HIV replication. Further deletion mapping demonstrated that expression of a 30 aa fragment resulted in decreased CXCR4 surface expression, upregulation of SDF-1 and inhibition of HIV replication.

RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum χ 2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.

Analysis of the VP1 capsid-coding sequences of the simian picornaviruses has suggested that baboon enterovirus (BaEV), SV19, SV43 and SV46 belong to the species Human enterovirus A (HEV-A) and SA5 belongs to HEV-B, whereas SV4/A2 plaque virus (two isolates of a single serotype), SV6 and N125/N203 (two isolates of a single serotype) appear to represent new species in the genus. We have further characterized by complete genomic sequencing the genetic relationships among the simian enteroviruses serotypes (BaEV, N125/N203, SA5, SV4/A2 plaque virus, SV6, SV19, SV43 and SV46) and to other enteroviruses. Phylogenetic and pairwise sequence relationships for the P1 region paralleled those of VP1 alone, and confirmed that SV4/A-2 plaque virus, SV6 and N125/N203 represent unique genetic clusters that probably correspond to three new species. However, sequence relationships in the P2 and P3 regions were quite different. In 2C, SV19, SV43 and SV46 remain clustered with the human viruses of HEV-A, but BaEV, SV6 and N125/N203 cluster together; in 3CD, SA5 (HEV-B) also joined this cluster. The 3′-non-translated region (NTR) sequences are highly conserved within each of the four human enterovirus species, but the 3′-NTRs of the simian enteroviruses are distinct from those of all human enteroviruses and generally distinct from one another. These results suggest that host species may have a significant influence on the evolution of enterovirus non-capsid sequences.

Semliki Forest virus (SFV) infection of the mouse provides a powerful model to study the pathogenesis of virus encephalitis. SFV and other alphavirus-based vector systems are increasingly used in biotechnology and medicine. This study analysed the strong susceptibility of this virus to type I interferon (IFN) responses. Following intraperitoneal infection of adult mice, SFV strain A7(74) was efficiently (100 %) neuroinvasive. In contrast, SFV4 was poorly (21 %) neuroinvasive. Upon entry into the brain, both viruses activated type I IFN responses. As determined by quantitative RT-PCR, activation of the IFN-α gene was proportional to virus RNA load. An intact type I IFN system was required for protection against both strains of SFV. IFN strongly curtailed virus spread in many cell types and in many tissues. In mice with an intact type I IFN system, infected cells were rarely observed and tissue tropism was difficult to determine. In the absence of a functional type I IFN system, the tropism and the potential for rapid and widespread infection of this virus was revealed. Virus infection was readily observed in the myocardium, endocardium, exocrine pancreas, adipose tissue, smooth muscle cells and in the brain in meningeal cells, ependymal cells and oligodendrocytes. In the brains of mice with and without type I IFN responses, virus infection of neurons remained rare and focal, indicating that the previously described restricted replication of SFV A7(74) in neurons is not mediated by type I IFN responses.

Reverse-genetic systems are often used to study different aspects of the viral life cycle. To date, three rescue systems have been developed for the family Bunyaviridae. These systems use T7 RNA polymerase, which is generally used in rescue systems for Mononegavirales. In the present study, we describe a rescue system for Akabane virus (family Bunyaviridae) that uses cDNAs and RNA polymerase I instead of T7 RNA polymerase. The utility of this system was demonstrated by the generation of a mutant with a deletion of the non-structural protein (NSs) on the S RNA segment. These results offer a new option for bunyavirus rescue.

Human metapneumovirus (hMPV) is a paramyxovirus that causes acute respiratory-tract infections in humans. The histopathological and immunological responses to hMPV infection in BALB/c mice immunized with inactivated hMPV were characterized. Animals were immunized intraperitoneally with PBS, supernatant from non-infected LLC-MK2 cells and from heat-inactivated influenza A- or hMPV-infected cells, all in incomplete Freund's adjuvant, or with heat-inactivated hMPV without adjuvant, and then infected intranasally with 108 TCID50 virus. Following infection, lung samples and bronchoalveolar lavages were collected for determination of viral titre and cytokine levels and for histopathological studies. On day 1, 26 % of mice immunized with inactivated hMPV and adjuvant died, compared with none in the other groups. There was more significant lung inflammation associated with eosinophilic infiltration, as well as increased levels of interleukin-4 (IL-4) and IL-5, in the bronchoalveolar lavages of mice immunized with hMPV alone or with the adjuvant. Mice from the last two groups had a 4–5 log10 decrease in their pulmonary viral titres compared with controls. Our data demonstrate the risks associated with immunization using inactivated hMPV in this animal model and that this aberrant response should be considered in the development of hMPV vaccines.

A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De λ3–CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be ‘identified’ as l1 ssRNAs, the individual nucleotides in these regions that ‘mark’ each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5′ 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5′ l1. RNA secondary-structure predictions for the 5′ 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5′ ssRNA predicted a common structure.

Middle Point orbivirus (MPOV) was isolated in 1998 from a healthy cow pastured at Beatrice Hill farm, Middle Point (formerly Coastal Plains Research Station), 50 km east of Darwin in Australia's Northern Territory. The isolate could not be identified by using conventional serological tests, and electron microscopy indicated that it belongs to the family Reoviridae, genus Orbivirus. Genetic sequencing of segments 2 and 3 revealed that this virus is related to Yunnan orbivirus, an orbivirus known only from China and not previously associated with a vertebrate host. A real-time RT-PCR test was developed to study the epidemiology of this virus in the field. Over 150 previously unidentified viruses isolated from cattle between 1994 and 2006 were positively identified as isolates of MPOV. Serology was used to demonstrate the development of antibody responses to MPOV in cattle from multiple locations across the Northern Territory.

In this study, characterization of the gag gene of small ruminant lentiviruses was carried out in Italian mixed flocks. The nearly complete gag gene was amplified and sequenced. Within genotype A, subtype A1 and a novel subtype, A8, were found in goats, and another novel subtype, A9, was found in both sheep and goats. Subtype B1 was found in both host species and subtype B2 was identified only in sheep. A novel, highly divergent sequence was obtained from goats in two epidemiologically related flocks and is proposed to represent a novel genotype, E. Major epitopes of matrix and capsid antigen were highly divergent, suggesting that serological identification of animals infected with genotype E may have been missed by using currently available diagnostic tests. A recombinant subunit ELISA, based on genotype E-specific epitopes, was developed and a third independent flock carrying this genotype was identified, based on serology.

We report the isolation, purification, genome-sequencing and characterization of a picorna-like virus from dead bees in Israel. Sequence analysis indicated that IAPV (Israeli acute paralysis virus) is a distinct dicistrovirus. It is most homologous to Kashmir bee virus and acute bee paralysis virus. The virus carries a 9487 nt RNA genome in positive orientation, with two open reading frames separated by an intergenic region, and its coat comprises four major proteins, the sizes of which suggest alternate processing of the polyprotein. IAPV virions also carry shorter, defective-interfering (DI)-like RNAs. Some of these RNAs are recombinants of different segments of IAPV RNA, some are recombinants of IAPV RNA and RNA from another dicistrovirus, and yet others are recombinants of IAPV and non-viral RNAs. In several of the DI-like RNAs, a sense-oriented fragment has recombined with its complement, forming hairpins and stem–loop structures. In previous reports, we have shown that potyviral and IAPV sequences are integrated into the genome of their respective hosts. The dynamics of information exchange between virus and host and the possible resistance-engendering mechanisms are discussed.

Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species. The A5 ORF of the AlHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [35S]GTPγS and reporter gene assays, we found that A5 is able to constitutively couple to α i-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AlHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AlHV-1 in MCF pathogenesis.

Cellular mismatch and base-excision repair machineries have been shown to be involved in Epstein–Barr Virus (EBV) lytic DNA replication. We report here that nucleotide-excision repair (NER) may also play an important role in EBV lytic DNA replication. Firstly, the EBV BGLF4 kinase interacts with xeroderma pigmentosum C (XPC), the critical DNA damage-recognition factor of NER, in yeast and in vitro, as demonstrated by yeast two-hybrid and glutathione S-transferase pull-down assays. Simultaneously, XPC was shown, by indirect immunofluorescence and co-immunoprecipitation assays, to interact and colocalize with BGLF4 in EBV-positive NA cells undergoing lytic viral replication. In addition, the efficiency of EBV DNA replication was reduced about 30–40 % by an XPC small interfering RNA. Expression of BGLF4 enhances cellular DNA-repair activity in p53-defective H1299/bcl2 cells in a host-cell reactivation assay. This enhancement was not observed in the XPC-mutant cell line XP4PA-SV unless complemented by ectopic XPC, suggesting that BGLF4 may stimulate DNA repair in an XPC-dependent manner. Overall, we suggest that the interaction of BGLF4 and XPC may be involved in DNA replication and repair and thereby enhance the efficiency of viral DNA replication.

Adenovirus infection subverts nucleolar structure and function. B23 is a nucleolar protein present in two isoforms (B23.1 and B23.2) and both isoforms have been identified as stimulatory factors for adenovirus DNA replication. Here, it is demonstrated that the two isoforms of B23, B23.1 and B23.2, interact and co-localize differently with viral DNA replication proteins pTP and DBP in adenovirus-infected cells. Thus, the mechanism by which the two proteins stimulate viral DNA replication is likely to differ. These data also demonstrate the importance of testing both isoforms of B23 for interactions with viral proteins and nucleic acids.

Chorioallantois vaccinia virus Ankara (CVA) is the parental virus of modified vaccinia virus Ankara (MVA), which was derived from CVA by more than 570 passages in chicken embryo fibroblasts (CEF). MVA became severely host-cell-restricted to avian cells and has strongly diminished virulence in mammalian hosts, while maintaining good immunogenicity. We determined the complete coding sequence of the parental CVA and mapped the exact positions of the six major deletions that emerged in the MVA genome. All six major deletions occurred in regions of the CVA genome where one or more truncated or fragmented open reading frames (ORFs) pre-existed. The CVA genome contains 229 ORFs of which 51 are fragments of full-length orthopoxvirus (OPV) genes, including fragmented orthologues of C9L and M1L (encoding two well-conserved ankyrin-like proteins), A39R (encoding a semaphorin-like protein) and A55R (encoding a kelch-like protein). Phylogenetic analysis demonstrated that MVA was most closely related to CVA, followed by the vaccinia virus (VACV) strain DUKE, a patient-derived isolate of the Dryvax vaccine virus. Loss or mutation of genes outside the six major deletions are assumed to contribute to the restricted host range phenotype of MVA. In support of this notion, deletions, insertions and non-synonymous mutations were found in 122 of the 195 ORFs remaining in MVA when compared with their CVA counterparts. Thus, detailed knowledge of the CVA genomic sequence is a prerequisite to further dissect the genetic basis of the MVA host range phenotype as well as the particular immunological properties of MVA.

Previous studies have suggested that hepatitis B virus (HBV) blocks expression of the alpha interferon (IFN-α)-inducible myeloid differential primary response protein (MyD88) gene. To study the molecular mechanism(s) of the inhibition of MyD88 expression by HBV, MyD88 promoter reporter plasmids and vectors expressing different HBV viral proteins were constructed. Co-transfection experiments showed that IFN-induced MyD88 promoter activity was inhibited by HBV polymerase expression in a dose-dependent manner and that the terminal protein (TP) domain of HBV polymerase was responsible for this antagonistic activity. Analysis of site mutants showed that the region targeted by the polymerase protein contained the signal transducer and activator of transcription (Stat) binding site. Chromatin immunoprecipitation analysis showed that the IFN-induced DNA-binding activity of Stat1 was affected. Further study demonstrated that the HBV polymerase protein inhibited the Stat1 nuclear translocation induced by IFN-α, but did not induce Stat1 degradation nor interfere with its phosphorylation. In addition, HBV polymerase could inhibit the transcriptional activity of other IFN-stimulated response element-driven promoters and the expression of interferon-stimulated genes (ISGs), such as Stat1 and ISG15. In summary, these results indicate that HBV polymerase is a general inhibitor of IFN signalling and can inhibit IFN-inducible MyD88 expression by inhibiting the activity of the MyD88 promoter through blocking the nuclear translocation of Stat1.

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Hepatitis B x antigen (HBxAg) contributes significantly to the pathogenesis of chronic infection and development of hepatocellular carcinoma. To discern some of its operative pathways, HepG2 cells were stably transduced with HBx or the bacterial chloramphenicol acetyltransferase (CAT) gene. Differential gene expression has previously revealed an upregulated gene, clone 7 (URG7), that conferred resistance to anti-Fas killing on HepG2X cells. Given that tumour necrosis factor alpha (TNFα) is also an important mediator of chronic hepatitis, and partially shares signalling with Fas, experiments were designed to test whether URG7 blocks TNFα killing of HepG2X cells. HepG2X cells expressing URG7 and HepG2 cells overexpressing URG7 in the absence of HBxAg were resistant to TNFα killing compared with HepG2CAT cells. URG7 small interfering RNA restored the sensitivity of HepG2X cells to TNFα killing. Killing was associated with the activation of caspases 3 and 8, suggesting that URG7 blocked these caspases. This resistance was also associated with activation of phosphoinositol 3-kinase/Akt. Given that Akt and HBxAg also activate β-catenin, experiments were designed to determine whether URG7 blocked apoptosis via activation of β-catenin. Both HBxAg and URG7 activated fragments of the β-catenin promoter, and also promoted expression of β-catenin target genes. Hence, URG7 inhibits TNFα-mediated killing by blocking one or more caspases in the apoptotic pathway and by activating phosphoinositol 3-kinase and β-catenin, thereby overriding the apoptotic signalling of TNFα. This suggests that URG7 helps to protect virus-infected hepatocytes during chronic hepatitis B virus infection.

Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.