- Volume 88, Issue 4, 2007
Volume 88, Issue 4, 2007
- Animal
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- RNA viruses
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Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors
Folding and assembly into complexes of some viral glycoproteins are exquisitely sensitive to endoplasmic reticulum (ER) α-glucosidase inhibition, which prevents the trimming of glucose from N-linked glycans. Derivatives of deoxynojirimycin (DNJ) iminosugars, which are potent α-glucosidase inhibitors, were shown to have antiviral activity against bovine viral diarrhea virus, a pestivirus related to hepatitis C virus (HCV). The aim of this study was to determine whether these inhibitors would affect HCV infectivity and to provide novel insights on their mechanism of action. The overall antiviral activity of glucosidase inhibitors was shown by using the two most relevant models currently available: the cell-culture model enabling complete replication of the HCV JFH1 strain in Huh7.5 cells, and infectious HCV pseudotyped particles (HCVpp) produced in HEK-293T cells that display functional E1–E2 glycoprotein complexes. By using the latter model, it is shown that the inhibition of α-glucosidases by iminosugars results in the misfolding and misassembly of HCV glycoprotein pre-budding complexes. This inhibition of the assembly of E1–E2 in the ER of transfected HEK-293T cells leads to a reduction in the incorporation of E1–E2 complexes into HCVpp. More importantly, it is demonstrated that the infectivity of HCVpp that are released under treatment is reduced and that this reduction in infectivity is due to the incorporation of misfolded envelope glycoproteins in secreted particles. These properties suggest the potential usefulness of DNJ derivatives in combating HCV infection.
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Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1
More LessThe E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276–286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330–347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.
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Expression of the alternative reading frame protein of Hepatitis C virus induces cytokines involved in hepatic injuries
Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mφ) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mφ and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CΔARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1β, whereas production of these chemokines with AdCΔARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1β in Mφ. Wild-type Core sequence, but not CΔARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42–44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.
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West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis
More LessUsing West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.
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A detailed mutagenesis study of flavivirus cross-reactive epitopes using West Nile virus-like particles
More LessHuman flavivirus infections elicit virus species-specific and cross-reactive immune responses. The flavivirus envelope (E) glycoprotein is the primary antigen inducing protective immunity; however, the presence of cross-reactive antibodies in human sera creates problems for serodiagnosis. Using a West Nile virus-like particle system, we performed mutagenesis across all three E protein functional domains to identify epitope determinants for a panel of monoclonal antibodies (mAbs) raised against different flaviviruses and exhibiting diverse patterns of cross-reactivity. Residues within the highly conserved fusion peptide were the only epitope determinants identified and were important not only for broadly cross-reactive mAbs recognizing all of the medically important flavivirus serocomplexes, but also for less-broad, complex-reactive mAbs. Moreover, different substitutions at specific fusion peptide residues produced highly variable effects on antibody reactivity and virus-like particle secretion. These results support and extend the conclusion that the fusion peptide region constitutes an immunodominant epitope stimulating antibodies with diverse patterns of cross-reactivity.
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In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein
Studies on the NS1 protein of flaviviruses have concluded that formation of a stable homodimer is required for virus replication. However, previous work has reported that substitution of a conserved proline by leucine at residue 250 in NS1 of Kunjin virus (KUNV) eliminated dimerization, but allowed virus replication to continue. To assess whether this substitution has similar effects on other flaviviruses, it was introduced into an infectious clone of Murray Valley encephalitis virus (MVEV). Consistent with studies of KUNV, the mutant virus (MVEVNS1-250Leu) produced high levels of monomeric NS1 and the NS1 homodimer could not be detected. In contrast, wild-type MVEV cultures contained predominantly dimeric NS1. Retarded virus growth in Vero cells and loss of neuroinvasiveness for weanling mice revealed further similarities between MVEVNS1-250Leu and the corresponding KUNV mutant. To confirm that the lack of detection of dimeric NS1 in mutant virus samples was not due to denaturation of unstable dimers during Western blotting, a mAb (2E3) specific for the MVEV NS1 homodimer was produced. When NS1 protein was fixed in situ in mammalian and arthropod cells infected with wild-type or mutant virus, 2E3 reacted strongly with the former, but not the latter. These results confirmed that Pro250 in NS1 is important for dimerization and that substitution of this residue by leucine represents a conserved marker of attenuation for viruses of the Japanese encephalitis virus serocomplex. The inability to detect dimeric NS1 in supernatant or cell monolayers of cultures productively infected with mutant virus also suggests that dimerization of the protein may not be essential for virus replication.
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Cross-reactive antibody responses to nsp1 and nsp2 of Porcine reproductive and respiratory syndrome virus
More LessPorcine reproductive and respiratory syndrome virus (PRRSV) non-structural proteins (nsps) play a key role in processing and maturation of the repertoire of structural and nsps of the virion, but little is known about the anti-nsp immune response. Here, it was hypothesized that pronounced antibody responses are generated to PRRSV nsp1 and nsp2, as they are present in infected cells and cytolytic infection releases viral proteins into interstitial spaces. Accordingly, nsp1 and nsp2 were cloned and expressed, and antibody responses in the sera of infected and vaccinated pigs were determined. Pigs mounted significant cross-reactive antibody responses that appeared equivalent to or greater than the response to nucleocapsid (N). Antibody reactivity to nsp1 and N was highly dependent on refolding of denatured proteins, suggesting that the porcine antibody response is directed primarily to conformational epitopes. The proteins reacted with sera from pigs infected with other PRRSV strains, indicating that multiple epitopes are conserved. Antibody responses to nsp1 and nsp2 were much higher than those to nsp4, which is encoded on the same RNA molecule and is equivalent in predicted antigenicity. These findings suggest either that nsp1 and nsp2 are highly immunogenic or that they are expressed at higher levels than nsp4 in PRRSV-infected cells, or both. Strong antibody responses to nsp1 and nsp2 may benefit the host by limiting potentially pathological consequences of viral protease activities encoded in these proteins that are released from dying cells. The identification of strain-specific antibody responses to a highly variable region of nsp2 may also provide the basis for immunoassays that differentiate serological responses of vaccines from field isolates.
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An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene
More LessThus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.
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PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells
More LessPreviously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genus Enterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genus Cardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Making use of subgenomic replicons, evidence is provided that PDTC inhibits replication of these two viruses by disturbing viral RNA synthesis. Furthermore, it is shown that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC. Finally, it is shown that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs. In CVB3-infected cells, PDTC interferes strongly with the proteolytic activity of 3CDpro, as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CDpro into 3Cpro and 3Dpol. In mengovirus-infected cells, however, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CDpro. Instead, PDTC caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary ‘break’ that normally occurs at the 2A–2B junction. Thus, PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different.
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Molecular analysis of the S glycoprotein gene of bovine coronaviruses isolated in Japan from 1999 to 2006
More LessIn total, 55 isolates of Bovine coronavirus (BCoV) were collected from cases of enteric and respiratory disease occurring between 1999 and 2006 in Japan. Phylogenetic analysis of the polymorphic region of the S glycoprotein gene of these isolates, together with those of other known strains, classified the BCoV strains and isolates into four clusters. Recent field isolates display distinctive genetic divergence from the prototype enteric BCoV strains – Mebus, Quebec, Kakegawa, F15 and LY138 – and have diverged in three different aspects over 8 years. These data suggested that the genetic divergence in the polymorphic region of the S glycoprotein has progressed considerably; thus, molecular analysis of this region should be useful in investigating the molecular epidemiology of BCoV. In addition, based on the differences in amino acids among the isolates, our study did not reveal the presence of certain genetic markers of pathogenicity and clinical symptoms in this polymorphic region.
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Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus
More LessAlphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.
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Mapping and structural analysis of B-cell epitopes on the morbillivirus nucleoprotein amino terminus
More LessBy analysing the antigenic structure of the morbillivirus nucleoprotein (N) using a competitive-binding assay of monoclonal antibodies (mAbs), six different antigenic sites were identified previously. By using Pepscan methodology complemented by analysis of truncated N proteins, a better characterization of five of these antigenic sites was provided: I, II, III, IV and VI. mAbs specific to Rinderpest virus, defining antigenic sites II, III and IV, and those common to four morbilliviruses, delineating sites I and VI, were analysed in the present study. It was found that all but one mapped to the same region, between aa 120 and 149 of N. However, the mAb 3-1 epitope was located in the carboxy-terminal region (aa 421–525). This result may indicate the high immunogenicity of the amino-terminal variable region, at least in the mouse. It was surprising that the epitope of mAb 33-4, antigenic site VI, which recognized all morbilliviruses so far tested, was located in one of the two non-conserved regions between morbillivirus N proteins. It is shown that the conserved amino acid motif 126EAD128----131F-------148EN149 is critical for epitope constitution and recognition.
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Measles virus M and F proteins associate with detergent-resistant membrane fractions and promote formation of virus-like particles
More LessAssembly and release of particles comprise a late step in virus–host cell interactions. Though it may share major biological properties with its orthologues in related viruses, trafficking and oligomerization of the matrix (M) protein of Measles virus (MV) and its relative contribution to assembly and budding of particles from particular host cells have not been addressed in more detail. Plasmid-driven expression of authentic and mutant M proteins revealed that the amino acid at position 89, an important adaptation determinant for growth of attenuated strains in Vero cells, influences the electrophoretic mobility but not the intracellular distribution of M proteins, nor their ability to oligomerize or migrate as a doublet band in SDS-PAGE. M proteins were found to co-float with detergent-resistant membrane fractions (DRM) and this was enhanced upon co-expression of the F protein. In contrast to their DRM association, the ability of M proteins to promote release of virus-like particles (VLPs) was not affected by the presence of F proteins, which on their own also efficiently promoted VLP production. Thus, DRM recruitment of MV F and M proteins and their ability to drive particle formation are not correlated.
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Experimental infection of macaques with human metapneumovirus induces transient protective immunity
Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a causative agent of acute respiratory-tract illness. Two main hMPV lineages circulate worldwide and reinfections occur frequently. It is unclear what level of protection is induced by natural hMPV infection, what the durability of this protection is and whether it differs for reinfection with homologous or heterologous viruses. Here, protective immunity in cynomolgus macaques at different time points after inoculation with molecularly cloned prototype viruses of the two main lineages of hMPV has been addressed. Animals received a homologous challenge at 4, 6 or 12 weeks after the primary infection. In addition, animals that had been inoculated three times within 10 weeks were challenged with homologous or heterologous virus 8 months later. Primary infection with 107 TCID50 resulted in virus shedding and induction of virus-neutralizing antibody responses, with higher titres against the homologous than the heterologous virus. Infections associated with virus shedding and seroconversion protected completely from homologous reinfection within 6 weeks, and partly at 12 weeks, after primary infection. Eight months later, protection had waned to virtually undetectable levels. This study demonstrates that experimental hMPV infection induces transient protective immunity.
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In vivo evidence for quasispecies distributions in the bovine respiratory syncytial virus genome
We analysed the genetic evolution of bovine respiratory syncytial virus (BRSV) isolate W2-00131, from its isolation in bovine turbinate (BT) cells to its inoculation in calves. Results showed that the BRSV genomic region encoding the highly variable glycoprotein G remained genetically stable after virus isolation and over 10 serial infections in BT cells, as well as following experimental inoculation in calves. This remarkable genetic stability led us to examine the mutant spectrum of several populations derived from this field isolate. Sequence analysis of molecular clones revealed an important genetic heterogeneity in the G-coding region of each population, with mutation frequencies ranging from 6.8 to 10.1×10−4 substitutions per nucleotide. The non-synonymous mutations of the mutant spectrum mapped preferentially within the two variable antigenic regions of the ectodomain or close to the highly conserved domain. These results suggest that BRSV populations may evolve as complex and dynamic mutant swarms, despite apparent genetic stability.
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Contribution of H7 haemagglutinin to amantadine resistance and infectivity of influenza virus
More LessIn the present study we determined the antiviral effect of amantadine against influenza A/Netherlands/219/03 (H7N7) virus in cell culture and in a mouse model. Amantadine at concentrations <100 μM failed to inhibit virus replication in Madin–Darby canine kidney (MDCK) cells. When orally administered to mice for 5 days, amantadine at 15 mg kg−1 day−1 did not protect animals against lethal challenge with H7N7 infection, and virus titres in mouse organs were not reduced. However, sequence analysis of the M2 protein revealed none of the mutations previously described as being associated with amantadine resistance. We used reverse genetics to generate viruses containing the haemagglutinin (HA) or M gene of A/Netherlands/219/03 virus to investigate the role of these genes in amantadine sensitivity. All recombinant viruses carrying the HA segment of A/Netherlands/219/03 (H7N7) virus were amantadine-resistant, regardless of the origin of their other genes. To study the role of fusion activity in the mechanism of drug resistance, we introduced the Gly23→Cys mutation in the H7 fusion peptide. This substitution resulted in a decrease of the pH of fusion and was also associated with reduced virus replication in both MDCK cells and mice, as compared to that of the wild-type virus. We suggest that H7 HA protein plays a role in amantadine resistance, although all HA amino acids that participate in drug resistance still remain to be characterized. Our finding reveals that sequence analysis of the transmembrane domain of M2 protein may not adequately identify all drug-resistant variants.
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Differential onset of apoptosis in influenza A virus H5N1- and H1N1-infected human blood macrophages
More LessPathogenesis of the highly pathogenic avian influenza virus A/Hong Kong/483/97 (H5N1/97) remains to be investigated. It was demonstrated recently that H5N1 dysregulation of proinflammatory cytokines in human macrophages is a p38-kinase-dependent process. The results indicated that macrophages may play a role in disease severity. To investigate cellular responses to H5N1 infection further, apoptosis and its related pathways were studied in primary blood macrophages. Here, it is shown that the H5N1/97 virus triggered apoptosis, including caspases and PARP activation, in infected macrophages with a delayed onset compared with H1N1 counterparts. Similar results were also found in human macrophages infected by precursors of the H5N1/97 virus. Thus, these results showed that the delay in apoptosis onset in macrophages infected by H5N1/97 and its related precursor subtypes may be a means for the pathogens to have longer survival in the cells; this may contribute to the pathogenesis of H5N1 disease in humans.
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A reverse-genetics system for Influenza A virus using T7 RNA polymerase
The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.
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Genetic variability of the M genome segment of clinical and environmental Toscana virus strains
Twenty-seven strains of Toscana virus, collected over a period of 23 years and isolated from several localities and from different hosts (humans, arthropods and a bat), were investigated by sequencing of a portion of the M genomic segment comprising the GN glycoprotein coding region. Sequence data indicated that the divergence among isolates ranged from 0 to 5.7 % at the nucleotide level and from 0 to 3.4 % at the amino acid level. Phylogenetic analysis revealed four main clusters. A close correspondence between viral strains and area/year of isolation could not be demonstrated, whilst co-circulation of different viral strains in the same area and in the same time period was observed for both patients and environmental viral isolates. Alignment of the deduced amino acid sequences and evolutionary analysis indicated that most of the sites along the gene may be invariable because of purifying and/or neutral selection.
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Isolation and characterization of hantavirus carried by Apodemus peninsulae in Jilin, China
To provide a better understanding of hantavirus epidemiology in China, Korean field mice (Apodemus peninsulae) and striped field mice (Apodemus agrarius) were captured in Jilin province, China, where haemorrhagic fever with renal syndrome (HFRS) is endemic. Hantavirus antigens were detected in eight of the 130 A. peninsulae individuals and in four of the 193 A. agrarius individuals by using an immunofluorescence assay. Partial S and M segments were amplified from all of the antigen-positive samples. Furthermore, two hantaviruses (CJAp89 and CJAp93) were isolated successfully in cell culture and the entire S and M segments were amplified from one of them (CJAp93). Phylogenetic analysis of these sequences (partial or complete) showed that hantaviruses carried by A. peninsulae and A. agrarius form two distinct lineages, although viruses carried by A. peninsulae are similar to those isolated previously from A. agrarius in China and from HFRS patients in Russia. However, the viruses detected in A. peninsulae in China are genetically different from those detected in A. peninsulae in other countries. These data suggest that A. peninsulae is also a natural host for HTNV in north-eastern China.
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Comparative analysis of the full genome sequence of European bat lyssavirus type 1 and type 2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the G–L 3′ non-translated region
More LessWe report the first full-length genomic sequences for European bat lyssavirus type-1 (EBLV-1) and type-2 (EBLV-2). The EBLV-1 genomic sequence was derived from a virus isolated from a serotine bat in Hamburg, Germany, in 1968 and the EBLV-2 sequence was derived from a virus isolate from a human case of rabies that occurred in Scotland in 2002. A long-distance PCR strategy was used to amplify the open reading frames (ORFs), followed by standard and modified RACE (rapid amplification of cDNA ends) techniques to amplify the 3′ and 5′ ends. The lengths of each complete viral genome for EBLV-1 and EBLV-2 were 11 966 and 11 930 base pairs, respectively, and follow the standard rhabdovirus genome organization of five viral proteins. Comparison with other lyssavirus sequences demonstrates variation in degrees of homology, with the genomic termini showing a high degree of complementarity. The nucleoprotein was the most conserved, both intra- and intergenotypically, followed by the polymerase (L), matrix and glyco- proteins, with the phosphoprotein being the most variable. In addition, we have shown that the two EBLVs utilize a conserved transcription termination and polyadenylation (TTP) motif, approximately 50 nt upstream of the L gene start codon. All available lyssavirus sequences to date, with the exception of Pasteur virus (PV) and PV-derived isolates, use the second TTP site. This observation may explain differences in pathogenicity between lyssavirus strains, dependent on the length of the untranslated region, which might affect transcriptional activity and RNA stability.
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‘2A-like’ and ‘shifty heptamer’ motifs in penaeid shrimp infectious myonecrosis virus, a monosegmented double-stranded RNA virus
More LessPenaeid shrimp infectious myonecrosis virus (IMNV) is a monosegmented double-stranded RNA virus that forms icosahedral virions and is tentatively assigned to the family Totiviridae. New examinations of the IMNV genome sequence revealed features not noted in the original report. These features include (i) two encoded ‘2A-like’ motifs, which are likely involved in open reading frame (ORF) 1 polyprotein ‘cleavage’; (ii) a 199 nt overlap between the end of ORF1 in frame 1 and the start of ORF2 in frame 3; and (iii) a ‘shifty heptamer’ motif and predicted RNA pseudoknot in the region of ORF1–ORF2 overlap, which probably allow ORF2 to be translated as a fusion with ORF1 by −1 ribosomal frameshifting. Features (ii) and (iii) bring the predicted ORF2 coding strategy of IMNV more in line with that of its closest phylogenetic relative, Giardia lamblia virus, as well as with that of several other members of the family Totiviridae including Saccharomyces cerevisiae virus L-A.
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Human MxA protein confers resistance to double-stranded RNA viruses of two virus families
More LessThe interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases and accumulates in the cytoplasm. MxA is a key component of the innate antiviral response and has previously been shown to inhibit several viruses with single-stranded RNA genomes of both polarities and a DNA virus. In addition, MxA also targets two double-stranded RNA viruses, Infectious bursal disease virus and a mammalian reovirus as shown in this study. Thus, the antiviral spectrum of human MxA is broader than hitherto suspected. Interestingly, virus growth was not affected in cells expressing MxA(E645R), a mutant form of MxA that showed antiviral activity against orthomyxoviruses.
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Envelope-specific T-helper and cytotoxic T-lymphocyte responses associated with protective immunity to equine infectious anemia virus
More LessEquine infectious anemia virus (EIAV) infection of horses provides a valuable model for examining the natural immunological control of lentivirus infection and disease and the mechanisms of protective and enhancing vaccine immunity. We have previously hypothesized that the EIAV envelope (Env) proteins gp90 and gp45 are major determinants of vaccine efficacy, and that the development of protective immunity by attenuated viral vaccines may be associated with the progressive redirection of immune responses from immunodominant, variable Env segments to immunorecessive, conserved Env sequences. Whilst the antibody-neutralization determinants of Env have been defined, there are to date no comprehensive analyses of the lymphoproliferative (T-helper, Th) and cytotoxic T-cell (CTL) epitopes of the EIAV Env proteins. Thus, in the current study, synthetic-peptide methodologies were used to define regions of EIAV Env associated with protective vaccine immunity in a panel of 12 horses inoculated with the attenuated EIAVD9 vaccine and two asymptomatic carrier horses infected experimentally with the virulent EIAVPV strain expressing the same Env protein as the vaccine strain. The results of these studies identified 17 broadly reactive Th peptides and six broadly reactive CTL peptides in the Env proteins of EIAV that were associated with protective immunity. Thus, these data provide for the first time a comprehensive mapping of EIAV Env-specific cellular regions that can be used to examine the development of protective immunity and to evaluate potential cellular immune determinants of protective immunity.
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- DNA viruses
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Soluble 3-O-sulfated heparan sulfate can trigger herpes simplex virus type 1 entry into resistant Chinese hamster ovary (CHO-K1) cells
Herpes simplex virus type 1 (HSV-1) interaction with glycoprotein D (gD) receptors facilitates virus entry into cells. Chinese hamster ovary (CHO-K1) cells lacking cellular receptors allow virus to attach, but not to enter, implying a role for receptors during the post-attachment (entry) phase of HSV-1 infection. Here, it is shown that the presence of soluble heparan sulfate (HS) modified by 3-O-sulfotransferase-3 (3-OST-3), but not by 3-OST-1, triggered HSV-1 entry into resistant CHO-K1 cells. It was further demonstrated that a CHO-K1 mutant deficient in glycosaminoglycan synthesis became susceptible to entry when spinoculated in the presence of 3-OST-3-modified soluble HS, indicating that the role of the gD receptor is to trigger entry rather than cell attachment. In separate experiments, 3-OST-3-modified soluble HS also triggered fusion of HSV-1 glycoprotein-expressing cells with CHO-K1 cells. Taken together, these results show that association of gD with cell surface-bound receptor is not essential for HSV-1 entry and spread.
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Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek's disease virus
More LessThe complete DNA sequence of the Marek's disease virus serotype 1 vaccine strain CVI988 was determined and consists of 178 311 bp with an overall gene organization identical to that of the oncogenic strains. In examining open reading frames (ORFs), nine differ between vaccine and oncogenic strains. A 177 bp insertion was identified in the overlapping genes encoding the Meq, RLORF6 and 23 kDa proteins of CVI988. Three ORFs are predicted to encode truncated proteins. One, designated 49.1, overlaps the gene encoding the large tegument protein UL36 and encodes a severely truncated protein of 34 aa. The others, ORF5.5/ORF75.91 and ORF3.0/78.0, located in the repeat regions (diploid), encode a previously unidentified ORF of 52 aa and a truncated version of the virus-encoded chemokine (vIL-8), respectively. Subtle genetic changes were identified in the two ORFs encoding tegument proteins UL36 and UL49. Only one diploid ORF (ORF6.2/ORF75.6) present in the genomes of the three virulent strains is absent in the CVI988-BAC genome. Seventy non-synonymous amino acid substitutions were identified that could differentiate CVI988-BAC from all three oncogenic strains collectively. Estimates of the non-synonymous to synonymous substitution ratio (ω) indicate that CVI988 ORFs are generally under purifying selection (ω<1), whereas UL39, UL49, UL50, RLORF6 and RLORF7 (Meq) appear to evolve under relaxed selective constraints. No CVI988 ORF was found to be under positive evolutionary selection (ω≫1).
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Transcriptional reactivation of murine cytomegalovirus ie gene expression by 5-aza-2′-deoxycytidine and trichostatin A in latently infected cells despite lack of methylation of the major immediate-early promoter
More LessWe have used a spleen explant model to investigate mechanisms of murine cytomegalovirus latency and reactivation. Induction of immediate-early (ie) gene expression occurs in explants after approximately 9 days in culture and virus reactivation follows induction of ie gene expression with kinetics similar to that of productive infection in vitro. This occurs independently of TNF receptor signalling. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A results in more rapid induction of ie gene expression and reactivation of virus. Despite these results, which suggest a role for DNA methylation in maintenance of viral latency, we find that the major immediate-early promoter/enhancer is not methylated in latently infected mice. Our results support the hypothesis that latency is maintained by epigenetic control of ie gene expression, and that induction of ie gene expression leads to reactivation of virus, but suggest that these are not controlled by DNA methylation.
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Adenovirus vector delivery stimulates natural killer cell recognition
We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery.
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Impact of a basement membrane-degrading protease on dissemination and secondary infection of Autographa californica multiple nucleopolyhedrovirus in Heliothis virescens (Fabricus)
More LessScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane (BM) during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus and triggers melanization and tissue fragmentation in infected larvae shortly before death. As BMs are a potential barrier to the spread of baculovirus secondary infection to other tissues in the host, this study tested the hypothesis that the rapid death of insects infected with AcMLF9.ScathL was caused by accelerated secondary infection resulting from the degradation of host BMs by ScathL. Viruses expressing catalytically active or inactive ScathL were used to examine the effects of ScathL activity on budded virus release into the haemocoel during infection, the production of polyhedra in infected larvae and the rate of infection of the gut, trachea, haemocytes, fat body and Malpighian tubules. It was concluded that the enhanced insecticidal efficacy of the recombinant baculovirus that expresses ScathL does not result from altered tissue tropism or accelerated systemic infection. Implications for the role of the BM as a barrier to baculovirus dissemination within the host insect are discussed.
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Sequence and organization of the Heliothis virescens ascovirus genome
More LessThe nucleotide sequence of the Heliothis virescens ascovirus (HvAV-3e) DNA genome was determined and characterized in this study. The circular genome consists of 186 262 bp, has a G+C content of 45.8 mol% and encodes 180 potential open reading frames (ORFs). Five unique homologous regions (hrs), 23 ‘baculovirus repeat ORFs' (bro) and genes encoding a caspase homologue and several enzymes involved in nucleotide replication and metabolism were found in the genome. Several ascovirus (AV)-, iridovirus- and baculovirus-homologous genes were identified. The genome is significantly larger than the recently sequenced genomes of Trichoplusia ni AV (TnAV-2c) and Spodoptera frugiperda AV (SfAV-1a). Gene-parity plots and overall similarity of ORFs indicate that HvAV-3e is related more closely to SfAV-1a than to TnAV-2c.
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- Plant
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A novel, multipartite, negative-strand RNA virus is associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.)
More LessFour RNAs from a new plant-pathogenic virus, which we have tentatively named European mountain ash ringspot-associated virus (EMARAV), were identified and sequenced completely. All four viral RNAs could be detected in previous double-stranded RNA preparations. RNA 1 (7040 nt) encodes a protein with similarity to the RNA-dependent RNA polymerase of different members of the Bunyaviridae, a family containing five genera with viruses infecting invertebrates, vertebrates and plants. RNA 2 (2335 nt) encodes a 75 kDa protein containing a conserved motif of the glycoprotein precursor of the genus Phlebovirus. Immunological detection indicated the presence of proteins with the expected size of the precursor and one of its processing products. The amino acid sequence of protein p3 (35 kDa) encoded by RNA 3 shows similarities to a putative nucleocapsid protein of two still unclassified plant viruses. The fourth viral RNA encodes a 27 kDa protein that has no significant homology to any known protein. As is typical for members of the family Bunyaviridae, the 5′ and 3′ ends of all viral RNAs are complementary, which allows the RNA to form a panhandle structure. Comparison of these sequences demonstrates a conserved terminal part of 13 nt, similar to that of the bunyaviral genus Orthobunyavirus. Despite the high agreement of the EMARAV genome with several characteristics of the family Bunyaviridae, there are a few features that make it difficult to allocate the virus to this group. It is therefore more likely that this plant pathogen belongs to a novel virus genus.
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Stability of recombinant plant viruses containing genes of unrelated plant viruses
More LessThe stability of hybrid plant viruses that might arise by recombination in transgenic plants was examined using hybrid viruses derived from the viral expression vectors potato virus X (PVX) and tobacco rattle virus (TRV). The potato virus Y (PVY) NIb and HCPro open reading frames (ORFs) were introduced into PVX to generate PVX-NIb and PVX-HCPro, while the PVY NIb ORF was introduced into a vector derived from TRV RNA2 to generate TRV-NIb. All three viruses were unstable and most of the progeny viruses had lost the inserted sequences between 2 and 4 weeks post-inoculation. There was some variation in the rate of loss of part or all of the inserted sequence and the number of plants containing the deleted viruses, depending on the sequence, the host (Nicotiana tabacum vs Nicotiana benthamiana) or the vector, although none of these factors was associated consistently with the preferential loss of the inserted sequences. PVX-NIb was unable to accumulate in NIb-transgenic tobacco resistant to infection by PVY and also showed loss of the NIb insert from PVX-NIb in some NIb-transgenic tobacco plants susceptible to infection by PVY. These data indicate that such hybrid viruses, formed in resistant transgenic plants from a transgene and an unrelated virus, would be at a selective disadvantage, first by being targeted by the resistance mechanism and second by not being competitive with the parental virus.
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- Other Agents
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Molecular analysis of bovine spongiform encephalopathy infection by cDNA arrays
More LessHere, the first cDNA array analysis of differential gene expression in bovine spongiform encephalopathy (BSE) is reported, using a spotted cDNA array platform representing nearly 17 000 mouse genes. Array analysis identified 296 gene candidates for differential expression in brain tissue from VM mice in late-stage infection with the 301V strain of BSE, compared with brain tissue from normal, age-matched VM mice. Real-time PCR confirmed differential expression of 25 of 31 genes analysed. Some of the genes identified by array analysis as being expressed differentially are associated with ubiquitin/proteasome function, lysosomal function, molecular chaperoning of protein folding or apoptosis. Other genes are involved in calcium ion binding/homeostasis, zinc ion binding/homeostasis or regulation of transcription. Principal-component analysis shows that the global gene-expression profiles of the BSE-infected samples have gene-expression signatures that are markedly different from, and completely non-overlapping with, those obtained from the normal controls.
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Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle
The dose–response of cattle exposed to the bovine spongiform encephalopathy (BSE) agent is an important component of modelling exposure risks for animals and humans and thereby, the modulation of surveillance and control strategies for BSE. In two experiments calves were dosed orally with a range of amounts of a pool of brainstems from BSE-affected cattle. Infectivity in the pool was determined by end-point titration in mice. Recipient cattle were monitored for clinical disease and, from the incidence of pathologically confirmed cases and their incubation periods (IPs), the attack rate and IP distribution according to dose were estimated. The dose at which 50 % of cattle would be clinically affected was estimated at 0.20 g brain material used in the experiment, with 95 % confidence intervals of 0.04–1.00 g. The IP was highly variable across all dose groups and followed a log-normal distribution, with decreasing mean as dose increased. There was no evidence of a threshold dose at which the probability of infection became vanishingly small, with 1/15 (7 %) of animals affected at the lowest dose (1 mg).
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Polymorphisms of the prion protein gene coding region in born-after-the-reinforced-ban (BARB) bovine spongiform encephalopathy cattle in Great Britain
Polymorphisms of the prion protein gene are associated with differing susceptibilities to transmissible spongiform encephalopathy diseases, as shown for variant Creutzfeldt–Jakob disease in humans and scrapie in sheep, but not yet in cattle. Imposition of control measures in the UK, including a reinforced ruminant feed ban in 1996, has led to a reduction in the incidence of bovine spongiform encephalopathy (BSE). BSE-affected cattle born after 1996 in Great Britain have been termed born-after-the-reinforced-ban (BARB) cases. In this study, the PrP gene coding region from 100 BARB BSE cases and 66 matched healthy-control cattle was sequenced to investigate whether this would reveal a genetic basis to their origin. Polymorphisms identified were not found to be associated with increased susceptibility to BSE in the BARB cases. Analysis of BARB cases grouped either by clinical status or by whether they formed an isolated or clustered case was also undertaken, but differences were not found to be significant.
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Progression of prion infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy challenge
More LessThe presence of BSE prion infectivity in asymptomatic cattle and its tissue distribution are important concerns for both human and veterinary health and food safety. In this work, a collection of tissues from asymptomatic cattle challenged orally with BSE and culled at 20, 24, 27, 30 and 33 months have been used to inoculate intracerebrally BoPrP-Tg110 mice expressing bovine PrP to assess their infectivity. Results demonstrate that BSE infectivity in asymptomatic cattle is essentially restricted to the nervous system, Peyer's patches and tonsils, as reported previously for terminally BSE-diseased cattle. BSE infectivity was detectable in Peyer's patches and tonsils at all time points analysed, but infectivity in nervous tissues (brainstem and sciatic nerve) was only detectable after 27 months from inoculation. Infectivity in brainstem increased markedly at 33 months after inoculation. All other investigated tissues or fluids (spleen, skeletal muscle, blood and urine) revealed no detectable infectivity throughout the time course studied.
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Immunological differences between susceptible and resistant sheep during the preclinical phase of scrapie infection
In order to investigate the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression were determined in susceptible and resistant Suffolk sheep in the preclinical phase of infection. At 6 months of age, 12 ARQ/ARQ (susceptible) and nine ARR/ARR (resistant) scrapie-free Suffolk lambs were challenged subcutaneously with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post-inoculation (p.i.) and serial bleeds were collected at monthly intervals for up to 1 year p.i. An indirect double-labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge, significantly more PrP+ cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge, significantly more CD8+ and γΔ + T cells were detected in the PBMCs of the resistant sheep. However, at both 14 and 180 days p.i, CD21+ cell expression was significantly higher in the lymph node preparations of the susceptible sheep. In contrast, more CD4+ cells were detected in the lymph nodes of the resistant sheep at both time points. It was concluded that significant differences in immune cell subsets and PrP expression occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the preclinical phase of infection.
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Discriminating between cellular and misfolded prion protein by using affinity to 9-aminoacridine compounds
Quinacrine and related 9-aminoacridine compounds are effective in eliminating the alternatively folded prion protein, termed PrPSc, from scrapie-infected cultured cells. Clinical evaluations of quinacrine for the treatment of human prion diseases are progressing in the absence of a clear understanding of the molecular mechanism by which prion replication is blocked. Here, insight into the mode of action of 9-aminoacridine compounds was sought by using a chemical proteomics approach to target identification. Cellular macromolecules that bind 9-aminoacridine ligands were affinity-purified from tissue lysates by using a 9-aminoacridine-functionalized solid-phase matrix. Although the 9-aminoacridine matrix was conformationally selective for PrPSc, it was inefficient: approximately 5 % of PrPSc was bound under conditions that did not support binding of the cellular isoform, PrPC. Our findings suggest that 9-aminoacridine compounds may reduce the PrPSc burden either by occluding epitopes necessary for templating on the surface of PrPSc or by altering the stability of PrPSc oligomers, where a one-to-one stoichiometry is not necessary.
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