- Volume 91, Issue 10, 2010

Hepatitis C virus (HCV) NS5A protein is phosphorylated on multiple residues; however, despite extensive study, the precise identity of these sites has not been determined unambiguously. In this study, we have used a combination of immunoprecipitation and mass spectrometry to identify these phosphorylation sites. This analysis revealed the presence of a major phosphorylated residue within NS5A from the genotype 1b Con1 isolate – serine 249 (serine 2221 in polyprotein numbering). However, mutation of this residue (or the corresponding threonine in the JFH-1 isolate) to either a phosphomimetic (aspartate) or a phosphoablative (alanine) residue resulted in no phenotype. We conclude that phosphorylation of this residue, in the context of a highly culture-adapted HCV genome, does not play a role in either viral RNA replication or virus assembly. It is possible that it might be important in an aspect of virus biology that is not recapitulated faithfully in the Huh-7 cell-culture system.

In this territory-wide molecular epidemiology study of picornaviruses, involving 6765 dead wild birds from 201 species in 50 families over a 12 month period, three novel picornaviruses, turdiviruses 1, 2 and 3 (TV1, TV2 and TV3), were identified from birds of different genera in the family Turdidae. In contrast to many other viruses in birds of the family Turdidae or viruses of the family Picornaviridae, TV1, TV2 and TV3 were found exclusively in the autumn and winter months. Two genomes each of TV1, TV2 and TV3 were sequenced. Regions P1, P2 and P3 of the three turdiviruses possessed, respectively, <40, <40 and <50 % amino acid identities with those of other picornaviruses. Moreover, P1, P2 and P3 of TV1 also possessed, respectively, <40, <40 and <50 % amino acid identities with those of TV2 and TV3. Phylogenetic analysis revealed that TV1, TV2 and TV3 were distantly related to members of the genus Kobuvirus. Among the three turdiviruses, TV2 and TV3 were always clustered together, with high bootstrap supports of 1000. The genomic features of TV2 and TV3 were also distinct from TV1, including lower G+C contents, shorter leader protein and a preference for codon sequence NNT rather than NNC for amino acids that can use either NNT or NNC as codons (P<0.001 by χ 2-test). Based on our results we propose two novel genera, Orthoturdivirus for TV1, and Paraturdivirus for TV2 and TV3, in the family Picornaviridae. The type of internal ribosomal entry site for TV1, TV2 and TV3 remains to be determined.

Norovirus-like particles were imaged using atomic force microscopy. The mechanical stability of the virus-like particles (VLPs) was probed by nanoindentation at pH values ranging from 2 to10. This range includes pH values of the natural environment during the life cycle of noroviruses. The resistance of VLPs to indentation was constant at acidic and neutral pH. The Young's modulus was of the order of 30 MPa. At basic pH the compliance of the capsid increased along with an increase in diameter. This specific pH-dependent mechanical response of the capsid may be related to mechanisms controlling uptake and release of the RNA during infection. Consecutive indentations with pressures ≤300 bar demonstrated the ability of the capsids to fully recover from deformations comparable with the size of the capsid. The capsids can be viewed as nanocontainers with an inbuilt self-repair mechanism. At pH 10 the capsids lost their stability and were irreversibly destroyed after one single indentation.

Several novel astroviruses have been recently discovered in humans and in other animals. Here, we report results from our surveillance of astroviruses in human and rodent faecal samples in Hong Kong. Classical human astroviruses (n=9) and a human MLB1 astrovirus were detected in human faecal samples (n=622). Novel astroviruses were detected from 1.6 % of the faecal samples of urban brown rat (Rattus norvegicus) (n=441), indicating the prevalence of astrovirus infection in rats might be much lower than that recently observed in bats. These rat astroviruses were phylogenetically related to recently discovered human astroviruses MLB1 and MLB2, suggesting that the MLB viruses and these novel rat astroviruses may share a common ancestor.

Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp that is classified in the genus Okavirus, family Roniviridae, in the order Nidovirales. Separation of virion proteins treated with peptide-N-glycosidase-F (PNGase-F) in SDS-polyacrylamide gels and the use of glycoprotein-specific staining methods indicated that the gp116 and gp64 envelope glycoproteins possess N-linked rather than O-linked glycans. Competitive binding inhibition of lectins with various oligosaccharide specificities indicated that glycans linked to gp64 are mannose-rich, whilst glycans linked to gp116 possess terminal N-acetylgalactosamine and N-acetylglucosamine in addition to terminal mannose-type sugars. Mass spectrometry analyses of peptides generated from YHV proteins before and after deglycosylation with PNGase-F, using combinations of the endoproteinases trypsin, Asp-N and Lys-C, confirmed occupancy of six of the seven potential N-linked glycosylation sites in gp116 and three of the four potential sites in gp64.

The non-structural protein NS2, also called nuclear export protein, of influenza A virus contains a leucine-rich nuclear-export signal that could guide viral ribonucleoproteins to cross the nuclear pore complex (NPC) and complete directional nucleocytoplasmic trafficking. In this study, human nucleoporin 98 (hNup98), an NPC protein, was identified as an NS2-binding protein by using yeast two-hybrid screening of a human cDNA library. Interaction between NS2 and hNup98 was confirmed in yeast and mammalian cells. Mapping tests further demonstrated that aa 22–53 in the N-terminal region of NS2 and the glycine-leucine-phenylalanine-glycine (GLFG) repeat domain (aa 1–511) of hNup98 are crucial for the interaction of these two proteins. Confocal microscopy showed that hNup98 could specifically recruit NS2 to the nucleoli and that this process was inhibited by leptomycin B, a specific inhibitor of human chromosomal region maintenance 1 protein. NS2 recruitment to the nucleoli was through the N-terminal GLFG repeat domain of hNup98, but not through the C-terminal domain. Moreover, influenza virus infection downregulated Nup98 levels significantly in 293T and Madin–Darby canine kidney cells. Overexpression of the GLFG repeat domain of hNup98 apparently inhibited virus propagation. Together, these findings reveal the interaction between hNup98 and NS2. The GLFG repeat domain of hNup98 might competitively inhibit the interaction between NS2 and endogenous hNup98, consequently inhibiting virus propagation.

Vietnam is one of the countries most affected by highly pathogenic H5N1 influenza A viruses. To evaluate the potential pathogenicity in mammals of H5N1 viruses isolated from humans in Vietnam, we determined the sequences of all eight genes of 22 human isolates collected between 2003 and 2008 and compared their virulence in mice. The isolates were classified into clade 1 and clade 2.3.4 and differed in pathogenicity for mice. Whilst lysine at position 627 of PB2 (PB2-627K) is a critical virulence determinant for clade 2.3.4 viruses, asparagine at position 701 of PB2 and other unknown virulence determinants appear to be involved in the high pathogenicity of clade 1 viruses, warranting further studies to determine the factors responsible for the high virulence of H5N1 viruses in mammals.

To investigate the prevalence and evolution of the H5 subtype highly pathogenic avian influenza (HPAI) viruses circulating in poultry in China during 2007–2009, five molecular epidemiological surveys were carried out. A total of 21 591 swab samples were collected, and from them 55 H5 HPAI viruses were isolated. None of the 55 viruses carried any known mutations, which can render the virus binding to human SAa2,6Gal receptors. The surveys indicated that live-bird markets, backyard flocks and slaughtering sites were at greater risk of being infected with the viruses during winter, and Clades 2.3.2, 2.3.4 and 7 of the viruses co-circulated in poultry in China during 2007–2009. Viruses within Clades 2.3.2 and 7 have become genetically distinguishable from the viruses isolated before 2007 and antigenically distinguishable from the vaccine strains used in China. Viruses within Clade 2.3.2 have been circulating widely in China and caused a new wave of cross-continental spreading from Asia to Europe.

The severity of respiratory syncytial virus (RSV) infections appears to differ with age in both humans and bovines. A primary RSV infection in naïve infants and in young calves runs a more severe course when they are 1–6 months old than in their first month of life. The relative lack of clinical signs in the first month of age may be due to high levels of maternally derived neutralizing antibodies or low exposure to infectious virus. This study examined whether age-dependent differences in the pathogenesis of bovine RSV (bRSV) between neonatal and young calves may be due to differences in age-dependent immunocompetence. To study the effect of age and immune parameters on bRSV disease in neonatal and young calves, neonatal (1-day-old) calves without maternally derived antibodies were infected experimentally with bRSV and the severity of disease and immune responses were evaluated in comparison with disease in similar 6-week-old infected calves. Neonatal calves had more extensive virus replication and lung consolidation, but lower pro-inflammatory [in particular tumour necrosis factor alpha (TNF-α)] responses, specific humoral immune responses, lung neutrophilic infiltration and clinical signs of disease than 6-week-old calves. The lack of correlation between virus replication and clinical signs suggests an important role of pro-inflammatory cytokines, in particular TNF-α, in the disease. The capacity to produce pro-inflammatory TNF-α appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.

Engineered therapeutic viruses provide an alternative method for treating infectious diseases, and mathematical models can clarify the system's dynamics underlying this type of therapy. In particular, this study developed models to evaluate the potential to contain human immunodeficiency virus type 1 (HIV-1) infection using a genetically engineered ‘hunter’ virus that kills HIV-1-infected cells. First, we constructed a novel model for understanding the progression of HIV infection that predicted the loss of the immune system's CD4+ T cells across time. Subsequently, it determined the effects of introducing hunter viruses in restoring cell population. The model implemented direct and indirect mechanisms by which HIV-1 may cause cell depletion and an immune response. Results suggest that the slow progression of HIV infection may result from a slowly decaying CTL immune response, leading to a limited but constant removal of uninfected CD4 resting cells through apoptosis – and from resting cell proliferation that reduces the rate of cell depletion over time. Importantly, results show that the hunter virus does restrain HIV infection and has the potential to allow major cell recovery to ‘functional’ levels. Further, the hunter virus persisted at a reduced HIV load and was effective either early or late in the infection. This study indicates that hunter viruses may halt the progression of the HIV infection by restoring and sustaining high CD4+ T-cell levels.

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9.5 kb long and contains a single ORF flanked by 5′- and 3′-UTRs and a naturally polyadenylated 3′ tail, with a genome organization typical of members of the family Iflaviridae. The two strains, labelled ‘Rothamsted’ and ‘Harpenden’, are 83 % identical at the nucleotide level (94 % identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations. The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was <2 %, with positive samples found only in England and Switzerland, in colonies with variable degrees of Varroa infestation.

The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65–VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.

Herpesviruses characteristically disseminate from immune hosts. Therefore in the context of natural infection, antibody neutralizes them poorly. Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to understand gammaherpesvirus neutralization. MuHV-4 virions blocked for cell binding by immune sera remain infectious for IgG-Fc receptor+ myeloid cells, so broadly neutralizing antibodies must target the virion fusion complex – glycoprotein B (gB) or gH/gL. While gB-specific neutralizing antibodies are rare, its domains I+II (gB-N) contain at least one potent neutralization epitope. Here, we tested whether immunization with recombinant gB presenting this epitope could induce neutralizing antibodies in naive mice and protect them against MuHV-4 challenge. Immunizing with the full-length gB extracellular domain induced a strong gB-specific antibody response and reduced MuHV-4 lytic replication but did not induce detectable neutralization. gB-N alone, which more selectively displayed pre-fusion epitopes including neutralization epitopes, also failed to induce neutralizing responses, and while viral lytic replication was again reduced this depended completely on IgG Fc receptors. gB and gB-N also boosted neutralizing responses in only a minority of carrier mice. Therefore, it appears that neutralizing epitopes on gB are intrinsically difficult for the immune response to target.

Gammaherpesviruses are archetypal pathogenic persistent viruses. The known human gammaherpesviruses (Epstein–Barr virus and Kaposi's sarcoma-associated herpesvirus) are host-specific and therefore lack a convenient in vivo infection model. This makes related animal gammaherpesviruses an important source of information. Infection by murid herpesvirus 4 (MuHV-4), a virus originally isolated from bank voles (Myodes glareolus), was studied here. MuHV-4 infection of inbred laboratory mouse strains (Mus musculus) is commonly used as a general model of gammaherpesvirus pathogenesis. However, MuHV-4 has not been isolated from house mice, and no systematic comparison has been made between experimental MuHV-4 infections of mice and bank voles. This study therefore characterized MuHV-4 (strain MHV-68) infection of bank voles through global luciferase imaging and classical virological methods. As in mice, intranasal virus inoculation led to productive replication in bank vole lungs, accompanied by massive cellular infiltrates. However, the extent of lytic virus replication was approximately 1000-fold lower in bank voles than in mice. Peak latency titres in lymphoid tissue were also lower, although latency was still established. Finally, virus transmission was tested between animals maintained in captivity. However, as observed in mice, MuHV-4 was not transmitted between voles under these conditions. In conclusion, this study revealed that, despite quantitative differences, replication and the latency sites of MuHV-4 are comparable in bank voles and mice. Therefore, it appears that, so far, Mus musculus represents a suitable host for studying gammaherpesvirus pathogenesis with MuHV-4. Establishing transmission conditions in captivity will be a vital step for further research in this field.

Latent membrane protein 2A (LMP2A) is expressed in most Epstein–Barr virus (EBV)-associated malignancies. Besides its roles in the maintenance of latent infection and epithelial-cell transformation, LMP2A could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence polymorphisms in LMP2A from northern Chinese EBV-associated gastric carcinoma patients, nasopharyngeal carcinoma patients and healthy donors were identified and compared with the prototype B95-8 strain. Four consistent mutations were detected in all isolates. Frequent mutations in the analysed sequences distinguished two and seven types of sequence variation in exon 1 and exons 2–8, respectively, with no consistent association shown between the genotyping of the two gene fragments. The immunoreceptor tyrosine-based activation motif and PY motif in the amino terminus were strictly conserved. Nine of the 16 identified CTL epitopes were affected by at least one point mutation, which may confer complexity to proposed immunotherapeutic approaches for EBV-associated malignancies. Most changed epitopes showed higher mutation rates in tumour isolates than in throat-washing samples from healthy donors, in accordance with the idea that virus strains can evade immune surveillance by altering amino acids within LMP epitopes. This first detailed investigation of sequence variations in the LMP2A gene reveals classifiable sequence polymorphisms in exon 1 and exons 2–8, and encourages further work on the impact of viral gene variations on tumour persistence and CTL-based immunotherapy.

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue, with a size equivalent to only 22 % of that of the largest orthologues. The present study focused on determining whether BoHV-4 ORF73 is a bona fide gene and investigating whether it is essential for latency, as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early polycistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by the wild type and revertant recombinants. Together, these results demonstrate that, despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease, such as post-weaning multisystemic wasting syndrome, which involves lymphocyte depletion. However, little is known about the molecular mechanisms of lymphoid depletion. To gain insight into the interaction between virus and host cells, microarrays were used to analyse changes in genomic expression in lymph nodes following PCV2 infection of pigs, together with negative controls. Total RNA was subjected to microarray analysis with an Affymetrix Porcine Genome Array GeneChip. Of the 23 256 pig genes arrayed on a chip, 160 genes showed altered expression after infection (upregulated, 64; downregulated, 96). The altered genomic expression of 18 selected genes was confirmed by quantitative real-time PCR. The expression changes of numerous genes involved in innate immune defence (TLR1, CD14 and CD180), immunosuppressed responses (FGL2 and GPNMB), pro-inflammatory signals (galectin-3) and fasting processes (ANGPTL-4) indicate that PCV2 has developed an intricate mechanism to cause immunosuppression, inflammatory cell infiltration and weight loss in pigs. The results of this study provide a basis for understanding the molecular pathogenesis of PCV2 infection.