- Volume 92, Issue 4, 2011

Host factors interacting with the dengue virus (DENV) 3′ UTR are involved in virus replication, but their roles remain poorly understood. We used RNA affinity capture and mass spectrometry to identify p100 as a host cellular protein associated with the DENV 3′ UTR. By using RNA immunoprecipitation and confocal immunofluorescence analysis we demonstrated an interaction between p100 and the 3′ UTR in DENV-infected cells. We identified the A4 region (the extensive stem–loop structure at the 3′ end) as the binding site of p100 by studying deletion mutants. p100 knockdown specifically reduced the levels of viral RNA and viral protein in DENV-infected cells. Furthermore, downregulation of p100 reduced the expression of a heterologously expressed luciferase–3′ UTR(DENV) mRNA in an A4-dependent manner, confirming the binding data and the effects of p100 knockdown on viral replication. These results provide evidence that p100 interacts with the 3′ UTR of DENV and is required for normal DENV replication.

Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.

The hepatitis C virus (HCV) p7 ion channel and non-structural protein 2 (NS2) are both required for efficient assembly and release of nascent virions, yet precisely how these proteins are able to influence this process is unclear. Here, we provide both biochemical and cell biological evidence for a functional interaction between p7 and NS2. We demonstrate that in the context of a genotype 1b subgenomic replicon the localization of NS2 is affected by the presence of an upstream p7 with its cognate signal peptide derived from the C terminus of E2 (SPp7). Immunofluorescence analysis revealed that the presence of SPp7 resulted in the targeting of NS2 to sites closely associated with viral replication complexes. In addition, biochemical analysis demonstrated that, in the presence of SPp7, a significant proportion of NS2 was found in a detergent (Triton X-100)-insoluble fraction, which also contained a marker of detergent resistant rafts. In contrast, in replicons lacking p7, NS2 was entirely detergent soluble and the altered localization was lost. Furthermore, we found that serine 168 within NS2 was required for its localization adjacent to replication complexes, but not for its accumulation in the detergent-insoluble fraction. NS2 physically interacted with NS5A and this interaction was dependent on both p7 and serine 168 within NS2. Mutational and pharmacological analyses demonstrated that these effects were not a consequence of p7 ion channel function, suggesting that p7 possesses an alternative function that may influence the coordination of virus genome replication and particle assembly.

West Nile virus (WNV) is a mosquito-borne flavivirus responsible for epidemics of febrile illness, meningitis, encephalitis and flaccid paralysis. WNV gains entry into host cells through endocytosis. The acid pH inside endosomes triggers rapid conformational rearrangements of the flavivirus envelope (E) glycoprotein that result in fusion of the endosomal membrane with the virion envelope. Conformational rearrangements of the E glycoprotein can be induced by acid exposure in solution in the absence of target membranes, thus causing a loss of infectivity. Following a genetic approach to study this process, a WNV mutant with increased resistance to acid-induced inactivation was isolated and its complete genome was sequenced. A single amino acid substitution, T70I, in the E glycoprotein was found to be responsible for the increased acid resistance, which was linked to an increase in the sensitivity of infection to the chemical rise of endosomal pH, suggesting that the mutant required a more acid pH inside the endosomes for fusion. No alterations in viral infection kinetics, plaque size or induced mortality rates in mice of the mutant were noted. However, by means of virus competition assays, a reduction in viral fitness under standard culture conditions was observed for the mutant. These results provide new evidence of the adaptive flexibility to environmental factors – pH variation in this case – of WNV populations. Implications of the T70I replacement on the E glycoprotein structure–function relationship are discussed.

The four major flavivirus clades are transmitted by mosquitoes, ticks, directly between vertebrates or directly between arthropods, respectively, but the molecular determinants of mode of transmission in flaviviruses are unknown. To assess the role of the UTRs in transmission, we generated chimeric genomes in which the 5′ UTR, capsid and/or 3′ UTR of mosquito-borne dengue virus serotype 4 (rDENV-4) were replaced, separately or in combination, with those of tick-borne Langat virus (rLGTV). None of the chimeric genomes yielded detectable virus following transfection. Replacement of the variable region (VR) in the rDENV-4 3′ UTR with that of rLGTV generated virus rDENV-4-rLGTswapVR, which showed lower replication than its wild-type parents in mammalian but not mosquito cells in culture and was able to infect mosquitoes in vivo. Neither rDENV-4 nor rDENV-4-rLGTswapVR could infect larval Ixodes scapularis ticks immersed in virus, while rLGTV was highly infectious via this route.

Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South African Territories (SAT) type viruses, which exist as distinct genetic and antigenic variants in different geographical regions. A cross-serotype chimeric virus, vKNP/SAT2, was engineered by replacing the external capsid-encoding region (1B-1D/2A) of an infectious cDNA clone of the SAT2 vaccine strain, ZIM/7/83, with that of SAT1 virus KNP/196/91. The vKNP/SAT2 virus exhibited comparable infection kinetics, virion stability and antigenic profiles to the KNP/196/91 parental virus, thus indicating that the functions provided by the capsid can be readily exchanged between serotypes. As these qualities are necessary for vaccine manufacturing, high titres of stable chimeric virus were obtained. Chemically inactivated vaccines, formulated as double-oil-in-water emulsions, were produced from intact 146S virion particles of both the chimeric and parental viruses. Inoculation of guinea pigs with the respective vaccines induced similar antibody responses. In order to show compliance with commercial vaccine requirements, the vaccines were evaluated in a full potency test. Pigs vaccinated with the chimeric vaccine produced neutralizing antibodies and showed protection against homologous FMDV challenge, albeit not to the same extent as for the vaccine prepared from the parental virus. These results provide support that chimeric vaccines containing the external capsid of field isolates can be successfully produced and that they induce protective immune responses in FMD host species.

This study analysed phylogenetic evidence of recombination in sequences of hepatitis A virus (HAV) available in international databases. Isolation of distinct recombinant HAV strains has been reported previously; however, the prevalence of natural recombination and its role in HAV genetics remains obscure. Analysis of full genome sequences revealed evidence of common intratypic recombination among the most prevalent subtypes, IA and IIIA. Many of the available complete sequences of these genotypes carried phylogenetic signs of recombination in all genomic regions without obvious hotspots. In addition, and in line with previous reports, recombination between subtypes IA and IB was detected. A dataset of 104 published HAV sequences for the VP1–2A and 3CD genomic regions was also analysed. Multiple instances of phylogenetic incompatibility were found among subtypes IA and IIIA. Three cases of recombination disrupted the phylogenetic grouping of subtype IA HAV strains isolated in Japan within less than 4 years, indicating common intratypic recombination in HAV. There were no signs of recombination between different HAV genotypes, despite the fact that co-circulation of genotypes IA and IIIA has commonly been reported in different parts of the world and many sequences in the sampling in this study originated from the same geographical region. These results indicate that there is reproductive isolation between genotypes of HAV, as exists between enterovirus species, and suggest that common intratypic recombination constrains the diversity within a genotype and maintains HAV genotypes as global gene pools.

Global epidemiological analysis is vital for implementing progressive regional foot-and-mouth disease control programmes. Here, we have generated VP1 region sequences for 55 Indian type A outbreak strains and have included complete VP1 sequences from 46 other countries to obtain a comprehensive global phylogeographical impression. A total of 26 regional genotypes within three continental topotypes, based on a 15 % nucleotide divergence cut-off criterion, could be identified. These genotypes correlated with distinct evolutionary lineages in the maximum-likelihood phylogeny. During the last decade, ten genotypes have been in circulation the world over and it was evident that no type A strain has transgressed the continental barriers during this period. A single genotype (genotype 18) within the Asia topotype has been circulating in India with neither any incursion nor any long distance movement of virus out of the country during the last ten years, although close genetic and epidemiological links between viruses from Bhutan and India were revealed.

Porcine reproductive and respiratory syndrome (PRRS) has a major negative economic impact on the swine industry worldwide. During the investigation of PRRS virus (PRRSV) in mainland China, European genotype (EU, type 1) PRRSV isolates were detected in swine herds both with and without clinical symptoms. Two complete genome sequences for Chinese type 1 PRRSV isolates were identified from viruses isolated from lung tissue and sera. The two viruses, designated BJEU06-1 and NMEU09-1, produced cytopathic effects in primary porcine alveolar macrophages but not in Marc-145 cells, and had a mean diameter of 55 nm, as measured by transmission electron microscopy . Comparative sequence analysis revealed that they shared 87.0–91.5 % and 58.0–58.2 % identity with the EU and North American genotype (NA, type 2) prototypic strains LV and VR-2332, respectively. Remarkably, these isolates, characterized by concomitant deletions within non-structural protein 2 (Nsp2) and ORF3 hypervariable regions, have never been described. Phylogenetic trees showed that all of the novel Chinese isolates of European genotype are in the pan-European subtype 1 that is predominant in Europe. However, they evolved from different ancestors. These novel viruses are predicted to be products of the divergent evolution of ancestor PRRSV isolates introduced from Europe. This is the first report of type 1 PRRSV wild isolates being in mainland China. Our findings confirm that the Chinese type 1 PRRSV isolates originated from diverse progenitors and the type 1 and type 2 PRRSV isolates, having different biological properties, have coexisted on the Chinese mainland for several years.

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >109 viral RNA copies μg−1 total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼105 RNA copies μg−1 total RNA) and increased >104-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

While performing a nationwide survey of hepatitis E virus (HEV) infection among 450 wild boars (Sus scrofa leucomystax) that had been captured in Japan between November 2005 and March 2010, we found 16 boars (3.6 %) with ongoing HEV infection: 11 had genotype 3 HEV, four had genotype 4 HEV and the remaining boar was infected with HEV of an unrecognized genotype (designated wbJOY_06). The entire wbJOY_06 genome was sequenced and was found to comprise 7246 nt excluding the poly(A) tail. The wbJOY_06 isolate was highly divergent from known genotype 1–4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits (n=145) by 22.6–27.7 %, rat HEV isolates (n=2) by 46.0–46.2 %, and avian HEV isolates (n=5) by 52.5–53.1 % over the entire genome. A Simplot analysis revealed no significant recombination between the existing HEV strains of genotypes 1–4. Therefore, we propose that the wbJOY_06 isolate is the first member of a previously unidentified genotype.

Infectious salmon anemia virus (ISAV) is an orthomyxovirus responsible for a significant disease of farmed Atlantic salmon. Fallowing and re-establishment of the Atlantic salmon farming industry in the Faroes following a recent devastating infectious salmon anaemia (ISA) disease epidemic provided a unique opportunity to study the risk of re-emergence of disease. Over 53 months, 2787 of 34 573 (8.1 %) apparently healthy Atlantic salmon analysed tested positive for ISAV by RT-PCR. Sequence analysis revealed the putative low-pathogenic ISAV-HPR0 subtype in all cases. Results demonstrated that ISAV-HPR0 appeared as a seasonal and transient infection without detectable ISA mortality or pathology. This finding, coupled to an apparent gill tropism of ISAV-HPR0, suggests ISAV-HPR0 causes a subclinical respiratory infection more like seasonal influenza, as opposed to the systemic infection and serious disease caused by highly pathogenic ISAV. The mean time before marine sites became infected was 7.7 months after transfer to seawater of the fish, suggesting a potentially unknown marine reservoir of infection. Sequence analysis identified two main subtypes of ISAV-HPR0 sequences, one of which showed close genetic association with ISAV isolates responsible for the disease outbreak in the Faroes. Thus ISAV-HPR0 might represent an ancestor of pathogenic variants and thus be a potential risk factor in the emergence of new strains of disease-causing ISAV. Our data, however, suggest that the risk of emergence of pathogenic ISAV variants from a reservoir of ISAV-HPR0 is low. This risk is probably being further reduced by practical management strategies adopted in the Faroes and aimed at reducing the potential for maintenance and adaptation of ISAV-HPR0.

Prior to the introduction of the 2009 pandemic H1N1 virus from humans into pigs, four phylogenetic clusters (α-, β-, γ- and δ) of the haemagglutinin (HA) gene from H1 influenza viruses could be found in US swine. Information regarding the antigenic relatedness of the H1 viruses was lacking due to the dynamic and variable nature of swine lineage H1. We characterized 12 H1 isolates from 2008 by using 454 genome-sequencing technology and phylogenetic analysis of all eight gene segments and by serological cross-reactivity in the haemagglutination inhibition (HI) assay. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. The gene segments from the 2009 pandemic H1N1 formed clusters separate from North American swine lineage viruses, suggesting progenitors of the pandemic virus were not present in US pigs immediately prior to 2009. Serological cross-reactivity paired with antigenic cartography demonstrated that the viruses in the different phylogenetic clusters are also antigenically divergent.

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100 % mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1β and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.

Pigs are thought to be the most suitable donor animal for xenotransplantation. However, pigs harbour potentially hazardous infectious agents, termed porcine endogenous retroviruses (PERVs), in their genome. In this study, we generated a mAb against PERV-B surface (SU) envelope protein (Env), designated KRT1. KRT1 binding was detected by an indirect immunofluorescence assay and flow cytometric analysis on cells infected with PERV-B. KRT1 neutralized PERV-B pseudotype virus and specifically recognized PERV-B SU Env, but not PERV-A SU Env by immunoblotting analysis. The peptide-ELISA revealed that KRT1 recognized a linear peptide sequence (ALEPPHNLPVP) residing in a proline-rich region that is one of the subdomains of SU Env. In conclusion, the KRT1 antibody will serve as a useful tool for the study of PERV-B and, more importantly, it may provide new protective strategies against PERV-B infection in xenotransplantation.

While RNA interference (RNAi) has been widely used to study rotavirus gene function in vitro, the potential therapeutic role for RNAi in vivo has not been explored. To this end, we constructed two recombinant lentiviral vectors containing short hairpin RNA (shRNA) against non-structural protein-4 (NSP4) of bovine rotavirus (BRV), RNAi-351 and RNAi-492. RNAi-351 and RNAi-492 strongly suppressed the transient expression of a FLAG-tagged NSP4 fusion protein in 293T cells. In BRV-susceptible MA104 cells, RNAi-492 more potently silenced NSP4 mRNA than RNAi-351 and combination of the two shRNAs almost completely silenced viral NSP4 gene expression. While 100 % of suckling mice exposed to BRV and control shRNA developed severe diarrhoea, no suckling mice exposed to BRV in the presence of RNAi-492 or a combination of RNAi-492/RNAi-351 developed severe diarrhoea, and only 20 and 3.3 % developed mild diarrhoea, respectively. In addition, RNAi-492 and RNAi-351 markedly abrogated rotaviral replication in MA104 cells and significantly inhibited BRV replication in mouse pups. These results indicated that shRNAs silencing NSP4 gene had substantial antiviral properties and inhibited replication of BRV in a sequence-specific manner that may have clinical application.

We have reported a novel bovine rotavirus, the AzuK-1 (G21P[29]) strain, isolated from an asymptomatic calf. We isolated another bovine rotavirus, the Dai-10 strain, bearing new G24P[33] genotypes, assigned by the Rotavirus Classification Working Group (RCWG), from an asymptomatic cow in Hyogo Prefecture, Japan in 2007. To gain an insight into the origins and evolution of these strains, we determined the complete ORF sequences of all 11 genes of the two strains. The NSP3 genes of both strains were confirmed to belong to a new NSP3 genotype, T9, by the RCWG. Genotype determination of AzuK-1 and Dai-10 strains revealed that eight gene segments of both strains possessed genotypes typically observed in bovine rotaviruses, with the exception of VP4, VP7 and NSP3 gene segments. Unexpectedly, phylogenetic analyses showed that VP6 and NSP2 gene segments of the AzuK-1 and Dai-10 strains were clustered with those of simian or canine/feline rotaviruses, rather than with those of bovine rotaviruses. These findings indicate the possibility that both strains originated by interspecies transmission and multiple reassortment events involving bovine, simian and canine/feline rotaviruses, resulting in the introduction of some genes into the genetic background of bovine rotaviruses.

Herpes simplex virus type 1 (HSV-1) is an important human pathogen and a leading cause of infectious blindness in the developed world. HSV-1 exploits heparan sulfate proteoglycans (HSPG) for attachment to cells. While the significance of heparan sulphate (HS) moieties in HSV-1 infection is well established, the role of specific proteoglycan core proteins in the infection process remains poorly understood. The objective of this study was to assess the roles of syndecan-1 and syndecan-2 core proteins in HSV-1 infection, both of which are expressed by many HSV-1 target cell types. Our results demonstrate that syndecan-1 and syndecan-2 gene silencing by RNA interference reduces HSV-1 entry, plaque formation and facilitates cell survival. Furthermore, HSV-1 infection increases syndecan-1 and syndecan-2 protein synthesis and a resultant increase in cell surface expression of HS. Our observations suggest that changes in syndecan-1 and syndecan-2 expression levels may be related to active viral infection. Taken together, our findings provide new insights into HSPG functions during HSV-1 entry and spread.

The link between Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) and Kaposi's sarcoma has been proven, but the transmission routes, especially in the heterosexual population, are not yet completely understood. To assess the intrafamilial patterns of transmission among first-degree relatives of Italian classic Kaposi's sarcoma (cKS) patients, KSHV seroprevalence and the presence of viral DNA in blood and saliva were evaluated in 18 families (32 cKS patients and 35 family members), comparing the results with those obtained in 200 elderly healthy controls without known exposure to KSHV. The KSHV genotype of variable region VR1 of the hypervariable ORF K1 gene was subsequently analysed in all KSHV-positive samples. The results showed that KSHV infection was significantly higher in relatives of cKS patients (11/35 cases) than in healthy controls (17/200 cases; P=0.001). The 11 infected relatives included spouses (n=3), siblings (n=2) and offspring (n=6) of the cKS patients; the same KSHV genotype was shared within the same family in the majority of cases (85 %), indicating the presence of person-to-person transmission within families. Viral DNA was mostly observed in the saliva of infected relatives (45.4 %); detection of DNA in blood was less frequent (27.3 %). Notably, KSHV DNA was present in saliva and/or blood of three KSHV-infected relatives with indeterminate or negative serostatus. Thus, the risk of KSHV infection is greatly enhanced within families of cKS patients, where close contacts (horizontal and/or sexual) can contribute to the spread of KSHV.