- Volume 91, Issue 10, 2010
Volume 91, Issue 10, 2010
- Review
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Proposals for the classification of human rhinovirus species C into genotypically assigned types
Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13 % divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing >10 % divergence in VP4/VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties.
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- Animal
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- RNA viruses
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Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal
Saint Louis encephalitis virus (SLEV), a member of the genus Flavivirus (family Flaviviridae), is an encephalitogenic arbovirus broadly distributed in the Americas. Phylogenetic analysis based on the full-length E gene sequences obtained for 30 Brazilian SLEV strains was performed using different methods including Bayesian and relaxed molecular clock approaches. A new genetic lineage was suggested, hereafter named genotype VIII, which co-circulates with the previously described genotype V in the Brazilian Amazon region. Genotypes II and III were restricted to São Paulo state (South-east Atlantic rainforest ecosystem). The analysis also suggested the emergence of an SLEV common ancestor between 1875 and 1973 (mean of 107 years ago), giving rise to two major genetic groups: genotype II, more prevalent in the North America, and a second group comprising the other genotypes (I and III–VIII), broadly dispersed throughout the Americas, suggesting that SLEV initially emerged in South America and spread to North America. In conclusion, the current study demonstrates the high genetic variability of SLEV and its geographical dispersion in Brazil and other New World countries.
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Identification of a novel phosphorylation site in hepatitis C virus NS5A
More LessHepatitis 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.
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Comparative analysis of six genome sequences of three novel picornaviruses, turdiviruses 1, 2 and 3, in dead wild birds, and proposal of two novel genera, Orthoturdivirus and Paraturdivirus, in the family Picornaviridae
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.
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Size and mechanical stability of norovirus capsids depend on pH: a nanoindentation study
More LessNorovirus-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.
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Detection of novel astroviruses in urban brown rats and previously known astroviruses in humans
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.
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Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp
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.
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Human cellular protein nucleoporin hNup98 interacts with influenza A virus NS2/nuclear export protein and overexpression of its GLFG repeat domain can inhibit virus propagation
More LessThe 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.
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Pathogenicity of highly pathogenic avian H5N1 influenza A viruses isolated from humans between 2003 and 2008 in northern Vietnam
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.
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Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007–2009
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.
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Age-dependent differences in the pathogenesis of bovine respiratory syncytial virus infections related to the development of natural immunocompetence
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.
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Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data
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.
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Effectiveness of a ‘hunter’ virus in controlling human immunodeficiency virus type 1 infection
More LessEngineered 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.
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Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.)
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.
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- DNA viruses
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Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells
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.
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Vaccination with murid herpesvirus-4 glycoprotein B reduces viral lytic replication but does not induce detectable virion neutralization
More LessHerpesviruses 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.
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Comparative study of murid gammaherpesvirus 4 infection in mice and in a natural host, bank voles
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.
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Widespread sequence variation in the Epstein–Barr virus latent membrane protein 2A gene among northern Chinese isolates
More LessLatent 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.
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Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo
More LessORF73 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.
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Genomic expression profiling in lymph nodes with lymphoid depletion from porcine circovirus 2-infected pigs
More LessPorcine 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.
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Hepatitis B doubly spliced protein, generated by a 2.2 kb doubly spliced hepatitis B virus RNA, is a pleiotropic activator protein mediating its effects via activator protein-1- and CCAAT/enhancer-binding protein-binding sites
More LessThe 2.2 kb doubly spliced defective hepatitis B virus (HBV) genome is frequently detected in the serum of patients with chronic hepatitis B. However, the biological significance of this type of defective genome is not well understood. In this study, expression of the hepatitis B doubly spliced protein (HBDSP) was confirmed from the 2.2 kb doubly spliced defective HBV genome, which was isolated and transfected into Huh-7 hepatoma cells. To explore the potential pathogenicity of HBDSP, hepatocellular proteins interacting with HBDSP were screened by a yeast two-hybrid assay. Unexpectedly, HBDSP could transactivate the GAL4-responsive element, and deletion mapping revealed that the fragment located between residues Leu-48 and Gln-75 of HBDSP was crucial for transactivation activity. In Huh-7 hepatoma cells, HBDSP localized predominantly to the cytoplasm and showed transactivating effects on the cytomegalovirus immediate-early promoter, simian virus 40 enhancer/promoter and HBV regulatory elements including the S1 promoter, S2 promoter, Enhancer I and core upstream regulatory sequences. Further studies revealed that the transactivating activities were mediated by activator protein-1- and CCAAT/enhancer-binding protein-binding sites. These findings suggest that HBDSP is a pleiotropic activator protein that can potentially serve as an HBV virulence factor.
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Prevalence and genetic diversity of adeno-associated viruses in bats from China
Bats are increasingly being recognized as important natural reservoirs of different viruses. Adeno-associated viruses (AAVs) are widely distributed in primates and their distribution in bats is unknown. In this study, a total of 370 faecal swab samples from 19 bat species were collected from various provinces of China and examined for the presence of AAVs. The mean prevalence rate was 22.4 % (83 positives out of 370 samples), ranging from 10 to 38.9 % among different bat species. The genome sequence spanning the entire rep–cap ORFs was determined from one chosen AAV-positive sample (designated BtAAV-YNM). Phylogenetic analysis of the entire rep–cap ORF coding sequences suggested that BtAAV-YNM is relatively distant to known primate AAVs, but phylogenetically closer to porcine AAV strain Po3. Further analysis of the partial cap ORF sequences of bat AAV samples (n=49) revealed a remarkably large genetic diversity, with an average pairwise nucleotide identity of only 84.3 %. Co-presence of multiple distinctive genotypes of bat AAV within an individual sample was also observed. These results demonstrated that diverse AAVs might be widely distributed in bat populations.
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Identification of bracovirus particle proteins and analysis of their transcript levels at the stage of virion formation
Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitic wasps; they replicate only in the calyx cells of a wasp's ovaries and are transferred at oviposition along with the parasitoid egg into the lepidopteran host. The DNA packaged in the viral particles encodes factors that manipulate the host's immune defences and development to benefit the parasitoid. PDVs are found in two subfamilies of ichneumonids (ichnoviruses) and in braconids of the microgastroid complex (bracoviruses). We recently showed that the latter derive from an ancestral nudivirus, as 24 nudivirus-related genes were identified in ovaries of two distantly related braconids at the stage of virion formation. Here, we present a comprehensive analysis of the viral particle proteins of the Chelonus inanitus bracovirus (CiBV). Proteins of purified CiBV particles were analysed by mass spectrometry and amino acid sequences matched to the existing ovarian-cDNA database. In addition, transcript quantities of identified genes were measured by quantitative real-time PCR in female pupae at the onset and peak of virion formation and at corresponding stages in male pupae. This combined approach allowed the identification of 44 CiBV particle proteins: 16 were nudivirus-related, three had similarity to ovarian proteins of another braconid, 11 had similarity to cellular proteins and 14 had no similarity to known proteins. The transcripts of all of them increased in female, but not male, pupae. These data confirm the important contribution of nudivirus genes but also indicate the presence of many lineage- or species-specific proteins possibly involved in the parasitoid–host interaction.
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- Plant
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A functional calcium-transporting ATPase encoded by chlorella viruses
Calcium-transporting ATPases (Ca2+ pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca2+ pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca2+ pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca2+ pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a 32P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca2+, thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca2+-transporting ATPase encoded by a virus.
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- Other Agents
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Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe
Variability of pathological phenotypes within classical sheep scrapie cases has been reported for some time, but in many instances it has been attributed to differences in the PRNP genotype of the host. To address this issue we have examined by immunohistochemistry (IHC) and Western blotting (WB) for the disease-associated form of the prion protein (PrPd), the brains of 23 sheep from five European countries, all of which were of the same ARQ/ARQ genotype. As a result of IHC examinations, sheep were distributed into five groups with different phenotypes and the groups were the same regardless of the scoring method used, ‘long’ or ‘short’ PrPd profiling. The groups made did not respond to the geographical origin of the cases and did not correlate with the vacuolar lesion profiles, which showed a high individual variability. Discriminatory IHC and WB methods coincided to detect a ‘CH1641-like’ case but otherwise correlated poorly in the classification of disease phenotypes. No other polymorphisms of the PRNP gene were found that could account for the pathological differences, except perhaps for a sheep from Spain with a mutation at codon 103 and a unique pathological phenotype. Preliminary evidence indicates that those different IHC phenotypes correlate with distinct biological properties on bioassay, suggesting that they are indicative of strain diversity. We therefore conclude that natural scrapie strains exist and that they can be revealed by detailed pathological examinations, which can be harmonized between laboratories to produce comparable results.
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Use of a preclinical test in the control of classical scrapie
More LessScrapie control in Great Britain (GB) was originally based on the National Scrapie Plan's Ram Genotyping scheme aimed at reducing the susceptibility of the national flock. The current official strategy to control scrapie in the national flock involves culling susceptible genotypes in individual, known affected flocks (compulsory scrapie flock scheme or CSFS). However, the recent development of preclinical test candidates means that a strategy based on disease detection may now be feasible. Here, a deterministic within-flock model was used to demonstrate that only large flocks with many home-bred ewes are likely to be a significant risk for flock-to-flock transmission of scrapie. For most other flocks, it was found that the CSFS could be replaced by a strategy using a currently available live test without excessive risk to other farmers, even if the proportion of susceptible genotypes in the flock is unusually large. Even for flocks that represent a high risk of harbouring a high prevalence of infection, there would be limited probability of onward transmission if scrapie is detected soon after disease introduction (typically less than 5 years). However, if detection of disease is delayed, the existing CSFS strategy may be the most appropriate control measure in these cases.
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Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein
Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilized in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterized.
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