- Volume 91, Issue 9, 2010

Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5′–3′ exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.

Dengue virus (DENV) is a mosquito-borne disease caused by four genetically and serologically related viruses termed DENV-1, -2, -3 and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2 and ED3. The ED3 domain contains DENV type-specific and DENV complex-reactive antigenic sites. To date, nearly all antigenic studies on the E protein have focused on DENV-2. In this study, the epitope recognized by a DENV-3 type-specific monoclonal antibody (mAb 14A4-8) was mapped to the DENV-3 ED3 domain using a combination of physical and biological techniques. Epitope mapping revealed that amino acid residues V305, L306, K308, E309, V310, K325, A329, G381 and I387 were critical for the binding of mAb 14A4-8 and amino acid residues T303, K307, K386, W389 and R391 were peripheral residues for this epitope. The location of the mAb 14A4-8 epitope overlaps with the DENV complex-reactive antigenic site in the DENV-3 ED3 domain.

Leukopenia and immunosuppression are characteristic clinical manifestations of classical swine fever and peripheral blood mononuclear cells (PBMCs) are major targets of classical swine fever virus. To investigate proteomic expression changes in swine PBMCs during lethal CSFV infection, proteins of PBMCs from five lethally CSFV-infected pigs were resolved by two-dimensional electrophoresis followed by mass spectrometry. Quantitative intensity analysis revealed that 66 protein spots showed altered expression, 44 of which were identified as 34 unique proteins by MALDI-TOF-MS/MS. Cellular functions of these proteins included cytoskeletal, energy metabolism, protein translation and processing, antioxidative stress, heat shock and blood clotting. Western blot analysis confirmed the upregulation of annexin A1 and downregulation of cofilin. Identification of these changed levels of expression provides an understanding at the molecular level of the response of in vivo target cells to CSFV infection and of the pathogenic mechanisms of leukopenia and immunosuppression induced by the virus.

Human enterovirus 71 (EV-71) is a cause of seasonal epidemics of hand, foot and mouth disease, and of less common but severe neurological manifestations. Uncertainty persists regarding the circulation of virus populations in several geographical areas and the timescale of their dissemination. We determined EV-71 sequences at loci 1D (VP1 capsid protein) and 3CD (non-structural proteins) in 86 strains recovered in Austria, France and Germany and performed an evolutionary genetic study of extant virus populations. Phylogenetic analyses positioned 78 of the 86 sequences within two clades among subgenogroups C1 and C2. A minor sequence cluster was assigned to subgenogroup C4. Analyses incorporating the available sequences estimated the substitution rate in genogroup C at 3.66×10−3 and 4.46×10−3 substitutions per site year−1 for loci 1D and 3CD, respectively, assuming a relaxed molecular-clock model for sequence evolution. Most of the ‘European’ strains belonged to clades C1b and C2b, which originated in 1994 [95 % confidence interval (CI), 1992.7−1995.8] and 2002 (95 % CI, 2001.6−2003.8), respectively. Estimates of divergence times for locus 3CD were consistent with those measured for locus 1D. Intertwining between clades representing EV-71 subgenogroups and clades corresponding to other enterovirus types (notably early coxsackievirus A prototype strains) in the 3CD phylogeny is highly indicative of ancestral recombination events. Incongruent phylogenetic patterns estimated for loci 1D and 3CD show that a single tree cannot model the epidemic history of circulating EV-71 populations. The evolutionary timescale of genogroup C estimated for both loci was measured only in decades, indicating recent dissemination.

Src family kinases (SFKs) are non-receptor tyrosine kinases that have been implicated as regulators of the inflammatory response. In this study, the role of SFK activation in the inflammatory response of macrophages to encephalomyocarditis virus (EMCV) infection was examined. Virus infection of macrophages stimulates the expression of cyclooxygenase-2 (COX-2), interleukin (IL)-1β and inducible nitric oxide synthase (iNOS). Inhibition of SFK attenuates EMCV-induced COX-2 expression and prostaglandin E2 production, iNOS expression and subsequent nitric oxide production, and IL-1β expression. EMCV-induced COX-2 expression requires the activation of nuclear factor-κB and the mitogen-activated protein kinase p38. Consistent with these previous findings, inhibition of SFKs attenuated the phosphorylation of p38 in response to EMCV infection, suggesting that SFKs may act upstream of p38. These findings provide evidence that SFK activation plays an active role in the regulation of inflammatory gene expression by virus-infected macrophages.

In 2006–2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.6–100.0 % (nsp2) and 97.8–100 % (ORF3) nucleotide identity and contained the unique insertions in nsp2 and ORF3, indicating that the EVA outbreaks in these states probably originated from the same strain of EAV. Sequence and phylogenetic analysis of several EAV isolates made following an EVA outbreak on another Quarter Horse farm in New Mexico in 2005 provided evidence that this outbreak may well have been the source of virus for the 2006–2007 occurrence of the disease. A virus isolate from an aborted fetus in Utah was shown to have a distinct neutralization phenotype compared with other isolates associated with the 2006–2007 EVA occurrence. Full-length genomic sequence analysis of 18 sequential isolates of EAV made from eight carrier stallions established that the virus evolved genetically during persistent infection, and the rate of genetic change varied between individual animals and the period of virus shedding.

To identify critical phenotypes that affect avian influenza virus transmission in chickens, we compared the transmissibility of three H5N1 highly pathogenic viruses of different pathogenicity in chickens by monitoring the exact time of death using wireless thermo-sensors. This study showed that, despite quick deaths, the most virulent H5N1 A/chicken/Yamaguchi/7/2004 transmitted quickly in chickens via contact and airborne routes. Intermediate virulent H5N1 A/chicken/Miyazaki/K11/2007 spread moderately and less virulent H5N1 A/duck/Yokohama/aq10/2003, causing severe clinical signs and a long period to death, spread slowly among the animals. The transmissibility was correlated with virus titres of oropharyngeal and cloacal swabs, and the time for swab virus titres to reach 50 % chicken infective dose affected the transmission speed. These results demonstrate that peak virus titres excreted and the time required for virus titres to reach a minimal chicken infectious dose may be the critical phenotypes influencing the transmissibility of highly pathogenicity avian influenza viruses in chickens.

Selection of an ideal sample is a vital element in early detection of influenza infection. Rapid identification of infectious individuals or animals is crucial not only for avian influenza virus (AIV) surveillance programmes, but also for treatment and containment strategies. This study used a combination of quantitative real-time RT-PCR with an internal positive control and a cell-titration system to examine the presence of virus in different samples during active experimental AIV infection and its persistence in the infected carcasses. Oropharyngeal/cloacal swabs as well as feather pulp and blood samples were collected from 15-day-old chicks infected with H7N1 highly pathogenic AIV (HPAIV) and the kinetics of virus shedding during active infection were evaluated. Additionally, several samples (muscle, skin, brain, feather pulp and oropharyngeal and cloacal swabs) were examined to assess the persistence of virus in the HPAIV-infected carcasses. Based on the results, feather pulp was found to be the best sample to detect and isolate HPAIV from infected chicks from 24 h after inoculation onwards. Kinetic studies on the persistence of virus in infected carcasses revealed that tissues such as muscle could potentially transmit infectious virus for 3 days post-mortem (p.m.), whilst other tissues such as skin, feather pulp and brain retained their infectivity for as long as 5–6 days p.m. at environmental temperature (22–23 °C). These results strongly favour feather as a useful sample for HPAIV diagnosis in infected chickens as well as in carcasses.

Triple-reassortant swine influenza viruses circulating in North American pigs contain the internal genes derived from swine (matrix, non-structural and nucleoprotein), human [polymerase basic 1 (PB1)] and avian (polymerase acidic and PB2) influenza viruses forming a constellation of genes that is well conserved and is called the triple-reassortant internal gene (TRIG) cassette. In contrast, the external genes [haemagglutinin (HA) and neuraminidase (NA)] are less conserved, reflecting multiple reassortant events that have produced viruses with different combinations of HA and NA genes. This study hypothesized that maintenance of the TRIG cassette confers a selective advantage to the virus. To test this hypothesis, pigs were co-infected with the triple-reassortant H3N2 A/Swine/Texas/4199-2/98 (Tx/98) and the classical H1N1 A/Swine/Iowa/15/1930 viruses and co-housed with a group of sentinel animals. This direct contact group was subsequently moved into contact with a second group of naïve animals. Four different subtypes (H1N1, H1N2, H3N1 and H3N2) of influenza virus were identified in bronchoalveolar lavage fluid collected from the lungs of the experimentally infected pigs, with most of the viruses containing TRIG from the Tx/98 virus. Interestingly, only the intact H3N2 Tx/98 virus was transmitted from the infected pigs to the direct-contact animals and from them to the second contact group of pigs. These results demonstrated that multiple reassortments can occur within a host; however, only specific gene constellations are readily transmissible. It was concluded that certain HA and NA gene pairs, in conjunction with the TRIG cassette, may have a competitive advantage over other combinations for transmission and maintenance in swine.

The minimal virus requirements for the generation of influenza virus-like particle (VLP) assembly and budding were reassessed. Using neuraminidase (NA) from the H5N1 and H1N1 subtypes, it was found that the expression of NA alone was sufficient to generate and release VLPs. Biochemical and functional characterization of the NA-containing VLPs demonstrated that they were morphologically similar to influenza virions. The NA oligomerization was comparable to that of the live virus, and the enzymic activity, whilst not required for the release of NA-VLPs, was preserved. Together, these findings indicate that NA plays a key role in virus budding and morphogenesis, and demonstrate that NA-VLPs represent a useful tool in influenza research.

RNA segment 8 (NS) of influenza A virus encodes two proteins. The NS1 protein is translated from the unspliced primary mRNA transcript, whereas the second protein encoded by this segment, NS2/NEP, is translated from a spliced mRNA. Splicing of influenza NS1 mRNA is thought to be regulated so that the levels of NS2 spliced transcripts are approximately 10 % of total NS mRNA. Regulation of splicing of the NS1 mRNA has been studied at length, and a number of often-contradictory control mechanisms have been proposed. In this study, we used 32P-labelled gene-specific primers to investigate influenza A NS1 mRNA splicing regulation. It was found that the efficiency of splicing of NS1 mRNA was maintained at similar levels in both virus infection and ribonucleoprotein-reconstitution assays, and NS2 mRNA comprised approximately 15 % of total NS mRNA in both assays. The effect of NS1 protein expression on the accumulation of viral NS2 mRNA and spliced cellular β-globin mRNA was analysed, and it was found that NS1 protein expression reduced spliced β-globin mRNA levels, but had no effect on the accumulation of NS2 mRNA. We conclude that the NS1 protein specifically inhibits the accumulation of cellular RNA polymerase II-driven mRNAs, but does not affect the splicing of its own viral NS1 mRNA.

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The interaction was initially established with native proteins by co-immunoprecipitating PUUV nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to bind to the predicted CT of Gc. The importance of Gn- and Gc-CTs to the recognition of RNP was further verified in pull-down assays using soluble peptides with binding capacity to both recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species, suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our previous results, we suggest that the complete hetero-oligomeric (Gn–Gc)4 spike complex of hantaviruses mediates the packaging of RNP into virions.

Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies suggest that pathogenic hantaviruses evade induction of innate antiviral responses, and this ability might determine the virulence in humans. Since reverse genetic systems are not available, in vitro reassortment is currently the only way to culture defined hantavirus variants. Here, we demonstrate for the first time the generation of a reassortant between a pathogenic Old World and a non-pathogenic New World hantavirus in vitro. The reassortant contained the glycoprotein coding M-segment derived from the pathogenic Puumala virus (PUUV) and the other genomic segments coding for the nucleocapsid protein and RNA-dependent RNA-polymerase from Prospect Hill virus (PHV), which is taken as non-pathogenic in humans. Exchange of the M-segment was confirmed by sequencing and virus neutralization test with PUUV-specific sera. Functional analysis of the reassortant and parental viruses revealed characteristic growth kinetics and innate immune responses as determined by expression analyses for lambda interferon and MxA, and by interferon-stimulated response element reporter gene studies. Consistent with previous studies with other pathogenic hantaviruses, PUUV elicited reduced innate responses if compared with PHV. In all these functional assays the reassortant revealed PHV-like phenotypes. Thus, neither the PUUV M-segment nor entry via specific M-segment directed pathways modulated the virus type-specific innate responses. Moreover, the data imply that this approach might be an option for production of attenuated viruses that could be used as vaccines against pathogenic hantaviruses.

Bats are natural reservoirs for the majority of lyssaviruses globally, and are unique among mammals in having exceptional sociality and longevity. Given these facets, and the recognized status of bats as reservoirs for rabies viruses (RABVs) in the Americas, individual bats may experience repeated exposure to RABV during their lifetime. Nevertheless, little information exists with regard to within-host infection dynamics and the role of immunological memory that may result from abortive RABV infection in bats. In this study, a cohort of big brown bats (Eptesicus fuscus) was infected intramuscularly in the left and right masseter muscles with varying doses [10−0.1–104.9 median mouse intracerebral lethal doses (MICLD50)] of an E. fuscus RABV variant isolated from a naturally infected big brown bat. Surviving bats were infected a second time at 175 days post-(primary) infection with a dose (103.9–104.9 MICLD50) of the same RABV variant. Surviving bats were infected a third time at either 175 or 305 days post-(secondary) infection with a dose (104.9 MICLD50) of the same RABV variant. When correcting for dose, similar mortality was observed following primary and secondary infection, but reduced mortality was observed following the third and last RABV challenge, despite infection with a high viral dose. Inducible RABV-neutralizing antibody titres post-infection were ephemeral among infected individuals, and dropped below levels of detection in several bats between subsequent infections. These results suggest that long-term repeated infection of bats may confer significant immunological memory and reduced susceptibility to RABV infection.

This study reports the first complete genome sequence of a caprine group A rotavirus (GAR) strain, GO34. The VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain GO34, detected in Bangladesh, were assigned to the G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genotypes, respectively. Strain GO34 was closely related to the VP4, VP6–7 and NSP4–5 genes of bovine GARs and the NSP1 gene of GO34 to an ovine GAR. Strain GO34 shared low nucleotide sequence identities (<90 %) with VP2–3 genes of other GARs, and was equally related to NSP3 genes of human, ruminant and camelid strains. The VP1, VP6 and NSP2 genes of strain GO34 also exhibited a close genetic relatedness to human G2, G6, G8 and G12 DS-1-like GARs, whereas the NSP1 of GO34 was also closely related to human G6P[14] strains. All these findings point to a common evolutionary origin of GO34 and bovine, ovine, antelope, guanaco and human G6P[14] GARs, although phylogenetically GO34 is not particularly closely related to any other rotavirus strains known to date.

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.

Infection of a cell by lentiviruses, such as human immunodeficiency virus type 1 or feline immunodeficiency virus, results in the formation of a reverse transcription complex, the pre-integration complex (PIC), where viral DNA is synthesized. In non-dividing cells, efficient nuclear translocation of the PIC requires the presence of the inner nuclear lamina protein emerin (EMD). Here, we demonstrate that EMD phosphorylation is induced early after infection in primary non-dividing cells. Furthermore, we demonstrate that EMD phosphorylation is dependent on virion-associated mitogen-activated protein kinase (MAPK). Specific inhibition of MAPK activity with kinase inhibitors markedly reduced EMD phosphorylation and resulted in decreased integration of the proviral DNA into chromatin. Similarly, when a MEK1 kinase-inactive mutant was expressed in virus-producer cells, virus-induced phosphorylation of EMD was impaired and viral integration reduced during the subsequent infection. Expression of constitutively active MEK1 kinase in producer cells did not result in modulation of EMD phosphorylation or viral integration during subsequent infection. These studies demonstrate that, in addition to phosphorylating components of the PICs at an early step of infection, virion-associated MAPK plays a role in facilitating cDNA integration after nuclear translocation through phosphorylation of target-cell EMD.

Secondary envelopment of herpes simplex virus type 1 has been demonstrated as taking place at the trans-Golgi network (TGN). The inner tegument proteins pUL36 and pUL37 and the envelope glycoproteins gD and gE are known to be important for secondary envelopment. We compared the cellular localizations of capsids from a virus mutant lacking the UL37 gene with those of a virus mutant lacking the genes encoding gD and gE. Although wild-type capsids accumulated at the TGN, capsids of the pUL37− mutant were distributed throughout the cytoplasm and showed no association with TGN-derived vesicles. This was in contrast to capsids from a gD−gE− mutant, which accumulated in the vicinity of TGN vesicles, but did not colocalize with them, suggesting that they were transported to the TGN but were unable to undergo envelopment. We conclude that the inner tegument protein pUL37 is required for directing capsids to the TGN, where secondary envelopment occurs.

VASP is an actin-regulatory protein that links signalling to remodelling of the cytoskeleton. We investigated the role of VASP during entry of herpes simplex viruses into epithelial MDCKII cells. As VASP functions are regulated by phosphorylations, the phosphorylation pattern was determined upon infection. Phosphorylated VASP decreased temporarily at 15 and 30 min after infection. The impact of phosphorylated VASP was addressed by overexpression of phosphomimetic VASP mutants. Our results revealed that phosphorylated VASP slightly reduced the number of infected cells. Expression studies with deletion mutants further indicated minor effects of VASP on infection efficiency, whereas RNA interference studies demonstrated that reduced VASP expression did not suppress infection. We conclude that VASP activities alone may contribute to herpes simplex virus infection to only a minor extent.