- Volume 81, Issue 11, 2000
Volume 81, Issue 11, 2000
- Review Article
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- Animal: RNA Viruses
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Resistance to Rift Valley fever virus in Rattus norvegicus: genetic variability within certain ‘inbred’ strains
More LessRift Valley fever virus (RVFV) is the causative agent of Rift Valley fever, a widespread disease of domestic animals and humans in sub-Saharan Africa. Laboratory rats have frequently been used as an animal model for studying the pathogenesis of Rift Valley fever. It is shown here that Lewis rats (LEW/mol) are susceptible to infection with RVFV, whereas Wistar–Furth (WF/mol) rats are resistant to RVFV infection. LEW/mol rats developed acute hepatitis and died after infection with RVFV strain ZH548, whereas WF/mol rats survived the infection. Cross-breeding of resistant WF/mol rats with susceptible LEW/mol rats demonstrated that resistance is segregated as a single dominant gene. Primary hepatocytes but not glial cells from WF/mol rats showed the resistant phenotype in cell culture, indicating that resistance was cell type-specific. Moreover, when cultured hepatocytes were stimulated with interferon (IFN) type I there was no indication of a regulatory role of IFN in the RVFV-resistance gene expression in WF/mol rats. Interestingly, previous reports have shown that LEW rats from a different breeding stock (LEW/mai) are resistant to RVFV infections, whereas WF/mai rats are susceptible. Thus, inbred rat strains seem to differ in virus susceptibility depending on their breeding histories. A better genetic characterization of inbred rat strains and a revision in nomenclature is needed to improve animal experimentation in the future.
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Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice
More LessThe continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98% homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.
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Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor
More LessFollowing binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis. Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus. We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C. We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced. Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I. Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations. We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented. Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible. Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium. These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.
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Role of CD4+ and CD8+ T cells in the prevention of measles virus-induced encephalitis in mice
Depending on their major histocompatibility complex (MHC) haplotype, inbred mouse strains are either resistant (H2-d, BALB/c), susceptible (H2-k, C3H) or partially resistant (H2-d×k, BaCF1) to intracerebral infection with the neurotropic rodent-adapted measles virus (MV) strain CAM/RBH. Here, mortality is demonstrated to be correlated directly with virus spread and virus replication in the CNS and to be inversely correlated with the activation of MV-specific T cells. Previously, it has been shown that primary CD4+ T cells alone are protective in the resistant background. In the susceptible background, CD4+ T cells acquire protective capacity after immunization with a newly defined CD4+ T cell epitope peptide. In the partially resistant mice, CD4+ T cells provide help for CD8+ T cells and protect in cooperation with them. It seems that the lytic capacity of CD8+ T cells is crucial in providing protection, as MV-specific Ld-restricted CD8+ T cells, which are highly lytic in vitro after transfer, protect naive animals against MV-induced encephalitis (MVE). In contrast, Kk-restricted CD8+ T cells with low lytic capacity do not protect. In the MVE model, CD4+ T cells are able to protect either alone (resistant mice), through cooperation with CD8+ T cells (intermediate susceptible) or after immunization as secondary T cells (susceptible mice). CD8+ T cells are able to protect alone after immunization if they are cytolytic. Thus, susceptibility and resistance depend upon the functional composition of CD4+ and CD8+ T cells governed by the MHC haplotype.
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Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans
More LessBinding of human respiratory syncytial virus (HRSV) to cultured cells was measured by flow cytometry. Using this assay and influenza virus as a control virus with a well-characterized receptor, a systematic search of cell surface molecules that might be implicated in HRSV binding was carried out. Treatment of cells with different enzymes or with other reagents suggested that heparin-like glycosaminoglycans (GAGs) were involved in attachment of HRSV, but not influenza virus, to host cells. This was further confirmed by a lack of binding of HRSV to CHO-K1 mutant cell lines deficient in glycosylation or GAGs biosynthesis and by an inhibition of binding after preincubation of virus with heparin and other GAGs. The degree of sulfation, more than the polysaccharide backbone of GAGs, seems to be critical for virus binding.
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Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup
More LessSequence analysis was performed of all or part of the genes encoding the fusion (F), polymerase (L) and attachment (G) proteins of two French non-A/non-B avian pneumovirus (APV) isolates (Fr/85/1 and Fr/85/2). The two isolates shared at least 99·7% nt and 99·0% aa sequence identity. Comparison with the F genes from subgroup A, subgroup B or Colorado APVs revealed nt and aa identities of 70·0–80·5% and 77·6–97·2%, respectively, with the L gene sharing 76·1% nt and 85·3% aa identity with that of a subgroup A isolate. The Fr/85/1 and Fr/85/2 G genes comprised 1185 nt, encoding a protein of 389 aa. Common features with subgroup A and subgroup B G proteins included an amino-terminal membrane anchor, a high serine and threonine content, conservation of cysteine residues and a single extracellular region of highly conserved sequence proposed to be the functional domain involved in virus attachment to cellular receptors. However, the Fr/85/1 and Fr/85/2 G sequences shared at best 56·6% nt and 31·2% aa identity with subgroup A and B APVs, whereas these isolates share 38% aa identity. Phylogenetic analysis of the F, G and L genes of pneumoviruses suggested that isolates Fr/85/1 and Fr/85/2 belong to a previously unrecognized APV subgroup, tentatively named D. G-based oligonucleotide primers were defined for the specific molecular identification of subgroup D. These are the first G protein sequences of non-A/non-B APVs to be determined.
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Equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic T lymphocytes from infected horses
Efficacious lentiviral vaccines designed to induce cytotoxic T lymphocytes (CTL) in outbred populations with a diverse repertoire of MHC class I molecules should contain or express multiple viral proteins. To determine the equine infectious anaemia virus (EIAV) proteins with epitopes most frequently recognized by CTL from seven horses infected for 0·5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in CTL assays. Gag p15 was recognized by CTL from 100% of these infected horses. p26 was recognized by CTL from 86%, SU and the middle third of Pol protein were each recognized by 43%, TM by 29%, and S2 by 14%. Based on these results, it is likely that a construct expressing the 359 amino acids constituting p15 and p26 would contain epitopes capable of stimulating CTL in most horses.
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Simian immunodeficiency virus resistance of macaques infused with interferon β-engineered lymphocytes
To test the in vivo anti-simian immunodeficiency virus (SIV) efficacy of interferon (IFN)-β-engineered lymphocytes, peripheral blood lymphocytes harvested from two uninfected macaques were transduced with a retroviral vector carrying a constitutively expressed IFN-β gene and reinfused, resulting in approximately 1 IFN-β-transduced cell out of 1000 circulating cells. The gene-modified cells were well tolerated and could be detected for at least 74 days without causing any apparent side effects. These two animals together with three untreated control macaques were then infected with SIVmac251. The two IFN-β-infused macaques are in good health, 478 days after infection, with a reduced plasma virus load and sustained numbers of CD4+ and CD8+ cells. Throughout the study, the proportion of IFN-β-transduced cells has been maintained. Of the three control macaques, two were characterized by a high plasma virus load and a decrease in CD4+ cells. One was moribund and was sacrificed 350 days after infection and the other now has fewer than 100 circulating CD4+ cells/ml. Unexpectedly, the third control macaque, which, like the two IFN-β-infused animals, had a low plasma virus load and a maintenance of CD4+ and CD8+ cell number, was characterized by a permanent level of serum IFN-β, of unknown origin, already present before SIV infection. Although no definite conclusion can be made in view of the limited number of animals, these data indicate that further exploration is warranted of an IFN-β-based anti-human immunodeficiency virus gene therapy.
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Cell-free synthesis of poliovirus: 14S subunits are the key intermediates in the encapsidation of poliovirus RNA
More LessIn a cell-free system of uninfected HeLa cells, programmed with poliovirus RNA, extraneous radiolabelled 14S subunits assembled with endogenous 14S subunits and interacted with newly synthesized RNA to form virions (160S). This result suggests that 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.
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Quantitative analysis of viral RNA kinetics in coxsackievirus B3-induced murine myocarditis: biphasic pattern of clearance following acute infection, with persistence of residual viral RNA throughout and beyond the inflammatory phase of disease
Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases of humans. To further understand the mechanism of enterovirus persistence and its relationship to organ pathology, virus infectivity and viral RNA kinetics in the heart and other target organs during acute and persistent phases of murine coxsackievirus B3 infection were investigated. These studies revealed a biphasic pattern of virus clearance. Thus, there was a rapid but incomplete clearance of viral RNA from the myocardium following the acute phase of virus replication, which paralleled the elimination of virus infectivity. The mean half-life of viral RNA between days 5 and 14 post-infection (p.i.) was 13·4 h. In contrast, a much slower rate of decline in viral RNA levels was observed during the post-infectious inflammatory phase of myocarditis. The mean half-life of viral RNA between days 14 and 90 p.i. was 14·1 days. Viral RNA persisted in the myocardium beyond the resolution of inflammation and was still detectable in a proportion of animals 90 days after infection. Clearance of viral RNA from other target organs occurred more rapidly, but the rate of clearance was largely independent of the level of viral RNA present during the acute phase of infection. Thus, while antiviral immune responses effectively eliminated infectious virus, clearance of residual viral RNA from the myocardium and other target organs was significantly delayed, despite a prolonged inflammatory response. These findings suggest that clearance of persistent enterovirus infection requires mechanisms different from those responsible for the elimination of virus infectivity.
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Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it
More LessThe Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H–I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.
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- Animal: DNA Viruses
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Blood clearance rates of adenovirus type 5 in mice
More LessPersistence of adenovirus type 5 in blood has implications for the pathogenicity of the virus infection and for the use of this virus in oncolysis and gene therapy. In this study, the kinetics of adenovirus clearance from blood in mice has been evaluated. After a single inoculation of concentrated virus into the vena cava, virus half-life was less than 2 min. Depletion of Kupffer cells (KC) resulted in increased viraemia. After tail-vein injection, virus and latex beads co-localized within KC. An important factor in clearance by KC is the negative charge of particles. Deletion of the hexon hypervariable region 1 acidic stretch decreased the negative charge of the virion but it did not increase blood persistence. Coating with PEG (‘PEGylation’) reduced the clearance rate but also reduced infectivity.
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Pyrogenicity of human adenoviruses
More LessHigh doses (>1·56×107 p.f.u.) of purified preparations of human adenovirus types 3, 5 and 8 exhibited definite pyrogenic activity when injected intravenously into rabbits. Complete pyrogenic tolerance was obtained not only with homologous types but also with heterologous types of adenovirus. No pyrogenic cross-tolerance was observed between each of these three adenovirus types and paramyxovirus pyrogen or bacterial lipopolysaccharide. Adenovirus pyrogenicity was retained after UV-inactivation, whereas it was inactivated by heating at 56 °C for 30 min. Adenovirus pyrogenicity was not neutralized by mixing with homologous type-specific antiserum but non-pyrogenic doses (107 p.f.u.) of adenovirus types 3, 5 and 8 became highly pyrogenic in the presence of type-specific antibodies at the optimal virus:antibody ratio. This enhanced pyrogenicity depended upon the virus–antibody complex. From these results, it is probable that the pyrogenic activity of the virus–antibody complex, rather than the pyrogenic activity of the virions, is the main contributor to fever in adenovirus infection under actual physiological conditions.
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Interaction with CBP/p300 enables the bovine papillomavirus type 1 E6 oncoprotein to downregulate CBP/p300-mediated transactivation by p53
The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6–CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.
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VP1 DNA sequences of JC and BK viruses detected in urine of systemic lupus erythematosus patients reveal no differences from strains expressed in normal individuals
More LessThe ubiquitous human polyomaviruses BK (BKV) and JC (JCV) persist with no adverse effects in immunocompetent individuals. Virus-induced pathogenesis has been linked to virus reactivation during impaired immune conditions. Previous studies have shown a significant difference between the VP1 DNA sequences of JCV obtained from control urine samples and those in progressive multifocal leukoencephalopathy brain samples. This difference could not be detected when comparing normal control urinary JCV DNA with DNA sequences from chronic progressive multiple sclerosis patients. Since BKV and JCV are readily activated in systemic lupus erythematosus (SLE) patients, the presence of specific strains, related to VP1 DNA sequences, was investigated in these patients. VP1 DNA sequences in 100 urine samples from 21 SLE patients and 75 urine samples from 75 healthy pregnant women were analysed and compared to previously reported sequences. The results show that the VP1 sequence profiles of JCV and BKV excreted by SLE patients do not differ significantly from those excreted by immunocompetent individuals. The European JCV subtypes 1A or 1B were represented among all JCV-positive urine specimens, while BKV VP1 sequences showed complete, or almost complete, identity with the MM or JL strains. Different urine samples from the same patient collected over a 1 year period were predominantly stable. BKV VP1 DNA in urine specimens from healthy pregnant women was only detected during the third trimester of their pregnancy. These results argue against SLE-specific JCV and BKV strains and suggest reactivation of the viruses rather than recurrent re-infections of patients with SLE.
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Murine gammaherpesvirus-68 infection of and persistence in the central nervous system
More LessMurine gammaherpesvirus-68 (MHV-68) was originally isolated from a bank vole by passage through mouse brain. Given its ability to replicate in mouse brain and its subsequent reisolation from trigeminal ganglia, it was originally considered to be an alphaherpesvirus. Molecular studies have now firmly established MHV-68 to be a gammaherpesvirus. Other gammaherpesviruses have been suggested to cause and in some cases shown to cause neurological disease. Given the isolation history of MHV-68, we have studied the ability of this virus to gain access to, to replicate in and to persist in the mouse CNS. Following intranasal inoculation the virus was not generally neuroinvasive. However, in mice with a deletion of the type-I interferon receptor gene, peripheral virus titres are higher and perivascular CNS infection was observed. There was no evidence of virus spread via olfactory routes. Direct intracerebral inoculation of virus was fatal with widespread infection and destruction predominantly of meningeal and ependymal cells. Hippocampal pyramidal neurons, oligodendrocytes, Bergmann glia cells in the cerebellar cortex and neural progenitor cells in the rostral migratory stream were also infected. A similar infection was observed in younger mice. CNS infection following virus reactivation was investigated by implantation of infected glial cells. Implantation into a brain ventricle led to widespread fatal infection, principally involving ependymal and meningeal cells. Implantation into the striatum resulted in a predominantly neuronal infection. Implantation of cells into mice transiently treated with the antiviral thionucleoside analogue 2′-deoxy-5-ethyl-β-4′-thiouridine resulted in survival with detection of virus-infected cells in the brain 1 year later.
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Latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8) binds ATF4/CREB2 and inhibits its transcriptional activation activity
More LessLatency-associated nuclear antigen (LANA), encoded by ORF 73 of Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus-8), may play an important role in the persistence of the viral episome by tethering it to host chromosomes during mitosis. It also has been suggested from its amino acid sequence features that LANA may have transcription-regulatory activity. Here, it is reported that LANA interacts with activating transcription factor (ATF) 4/cAMP response element-binding protein (CREB) 2, a member of the ATF/CREB family of transcription factors, and represses the transcriptional activation activity of ATF4/CREB2. Repression by LANA is independent of the DNA-binding ability of ATF4/CREB2, since LANA also represses transactivation of ATF4/CREB2 fused to the GAL4 DNA-binding domain and does not affect the DNA-binding ability of ATF4/CREB2 in an electrophoretic mobility shift assay. The putative leucine zipper domain of LANA is required for binding to the relatively conserved basic region/leucine zipper domain (bZIP) of ATF4/CREB2, suggesting that the interaction may involve leucine zipper dimerization.
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Characterization of the herpesvirus saimiri ORF73 gene product
The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 encoded protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localizes with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, the expression of HVS ORF73 in a stably transduced human carcinoma cell line, where the HVS genome persists as a non-integrated circular episome, has recently been shown. In this report, the characterization of the HVS ORF73 protein and the mapping of its functional domains are described. The results suggest that the HVS ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the amino terminus contains two functional nuclear localization signals, whereas the carboxy terminus is required for the distinctive speckled nuclear distribution pattern as observed with both the HVS and KSHV ORF73 proteins.
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The antigenic domain 1 of human cytomegalovirus glycoprotein B contains an intramolecular disulphide bond
More LessGlycoprotein B (gB, gpUL55) is the major antigen recognized by the neutralizing humoral immune response against human cytomegalovirus (HCMV). The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids (aa 552–635) for antibody binding. In this study, the structural requirements for antibody binding to AD-1 have been determined. The domain was expressed in prokaryotic and eukaryotic systems and analysed in immunoblots under reducing and non-reducing conditions. In addition, AD-1 was purified in an immunologically active form and the concentration of sulphydryl groups was determined. The data clearly show that the only form that is recognized by antibodies is a disulphide-linked monomer of AD-1. The disulphide bond is formed between cysteines at amino acid positions 573 and 610 of gB.
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The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-γ
More LessTo study the biological relevance of using bovine herpesvirus-1 (BHV-1) as a vector for expressing cytokines, a BHV-1 virus that expressed bovine interferon-γ (IFN-γ) was constructed. This recombinant virus (BHV-1/IFNγ) was then used to infect the natural host in a respiratory disease model. In vitro characterization of the recombinant interferon-γ confirmed that the cytokine expressed in BHV-1-infected cells was biologically active. The in vivo effects of the recombinant IFN-γ were then analysed during a primary infection and after reactivation of a latent infection. During the primary infection, similar body temperature, clinical responses and virus shedding were observed for calves infected with either recombinant BHV-1/IFNγ or parental gC−/LacZ+ virus. An analysis of cellular and humoral responses did not reveal any significant immunomodulation by BHV-1/IFNγ during the primary infection. The stability and activity of recombinant IFN-γ was also analysed following the establishment of a latent infection. The presence of recombinant IFN-γ did not significantly alter virus shedding following reactivation. The isolation of reactivated BHV-1/IFNγ virus confirmed that a functional IFN-γ gene was retained during latency. Thus, herpesviruses may provide virus vectors that retain functional genes during latency and recrudescence.
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Vesicular stomatitis virus and pseudorabies virus induce a vig1/cig5 homologue in mouse dendritic cells via different pathways
The homologous genes vig1 and cig5 were identified by differential display PCR as virus-induced genes in rainbow trout and humans, respectively. These genes are significantly related to sequences required for the biosynthesis of metal cofactors, but their function remains unknown. In this study, it is shown that the mouse homologue of vig1/cig5 was induced by vesicular stomatitis virus (VSV) and pseudorabies virus (PrV) in mouse spleen cells. Among a collection of cell lines from dendritic, myeloid, lymphoid or fibroblast lineages, only the dendritic cell line, D2SC1, showed expression of mvig after virus infection. This dendritic restriction was confirmed by our finding that mvig was also induced by both VSV and PrV in CD11c++ spleen cells, separated by magnetic purification or derived from bone marrow precursor cells. Similar to the fish rhabdovirus viral haemorrhagic septicaemia virus in trout cells, VSV directly induced mvig in the dendritic cell line D2SC1, but the PrV-mediated induction required the integrity of the interferon pathway. This result indicates that mvig is interferon-inducible like its fish and human homologues. Furthermore, mvig was also induced by LPS in bone marrow-derived cells. Thus, mvig expression seems to correlate with an activated state of dendritic cells subjected to different pathogen-associated stimuli.
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- Insect
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The inhibitors of apoptosis of Epiphyas postvittana nucleopolyhedrovirus
More LessIn this study, four inhibitor of apoptosis genes (iaps) in the genome of Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) that are homologous to iap-1, iap-2, iap-3 and iap-4 genes of other baculoviruses have been identified. All four iap genes were sequenced and the iap-1 and iap-2 genes were shown to be functional inhibitors of apoptosis. The iap-1, iap-2 and iap-3 genes contain two baculovirus apoptosis inhibitor repeat motifs and a C3HC4 RING finger-like motif. The activity of the iap genes was tested by transient expression in Spodoptera frugiperda (Sf-21) cells treated with the apoptosis-inducing agents actinomycin D, cycloheximide, anisomycin, tumour necrosis factor-α and UV light. The iap-2 gene prevented apoptosis induced by all agents tested, indicating activity towards a conserved component(s) of multiple apoptotic pathways. However, the iap-2 gene was unable to function in the absence of a gene immediately upstream of iap-2 that has homology to the orf69 gene of Autographa californica MNPV. The use of a CMV promoter rescued the apoptosis inhibition activity of the iap-2 gene, indicating that the upstream orf69 homologue is associated with expression of iap-2. The iap-1 gene was able to delay the onset of apoptosis caused by all of the induction agents tested but, unlike iap-2, was unable to prevent the development of an apoptotic response upon prolonged exposure of cells to the apoptosis induction agents. No anti-apoptotic activity was observed for the iap-3 and iap-4 genes of EppoMNPV.
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- Plant
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Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro
More LessTomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q423/G and Q620/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.
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Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus)
The polyprotein of Cocksfoot mottle virus (CfMV; genus Sobemovirus) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a −1 ribosomal frameshifting mechanism. In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.
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Mechanical transmission of Potato leafroll virus
More LessLike typical luteoviruses, Potato leafroll virus (PLRV) cannot be transmitted mechanically by rubbing plants with solutions containing virus particles. However, PLRV was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with Pea enation mosaic virus-2 (PEMV-2). Unlike the asymptomatic infections induced by either virus alone, double infections in Nicotiana benthamiana induced necrotic symptoms with some line patterning and vein yellowing. Infective PLRV was recovered from a purified virus preparation by inoculating plants mechanically with purified virus particles mixed with PEMV-2. Similarly, Beet mild yellowing virus was readily transmitted mechanically from mixtures containing PEMV-2. PLRV was also transmissible from mixtures made with extracts of plants infected with Groundnut rosette virus, although less efficiently than from mixtures containing PEMV-2. This novel means of transmitting PLRV, and perhaps other poleroviruses, should prove very useful in a number of fields of luteovirus research.
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Natural recombination between Tomato yellow leaf curl virus-Is and Tomato leaf curl virus
More LessThe complete genome sequences (2791 and 2793 nt) of isolates of Tomato yellow leaf curl virus-Is (TYLCV-Is) from Spain (SP72/97) and Portugal (Port2/95) were determined. These isolates are closely related to TYLCV-Is isolates reported in Japan (Japan-A and Japan-S) and Israel (Israel/Mild). Comparison of all sequenced isolates of TYLCV-Is showed that part of the genome comprising the intergenic region and the 5′-end of the rep gene of the Iran and Israel isolates was not closely related to that of other isolates. Phylogenetic analyses suggest that the Israel and Iran isolates may have chimeric genomes that have arisen by recombination between TYLCV-Is-like and tomato leaf curl virus (ToLCV)-like ancestors. The TYLCV-Is donors of the Iran and the Israel genomes were closely related to each other and to other known TYLCV-Is isolates. However, the ToLCV donors differed from each other, although both were related to ToLCV isolates from India (Bangalore-2 and Bangalore-4).
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- Other Agents
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PrPC expression in the peripheral nervous system is a determinant of prion neuroinvasion
More LessTransmissible spongiform encephalopathies are often propagated by extracerebral inoculation. The mechanism of spread from peripheral portals of entry to the central nervous system (neuroinvasion) is complex: while lymphatic organs typically show early accumulation of prions, and B-cells and follicular dendritic cells are required for efficient neuroinvasion, actual entry into the central nervous system occurs probably via peripheral nerves and may utilize a PrPC-dependent mechanism. This study shows that transgenic mice overexpressing PrPC undergo rapid and efficient neuroinvasion upon intranerval and footpad inoculation of prions. These mice exhibited deposition of the pathological isoform of the prion protein (PrPSc) and infectivity in specific portions of the central and peripheral sensory pathways, but almost no splenic PrPSc accumulation. In contrast, wild-type mice always accumulated splenic PrPSc, and had widespread deposition of PrPSc throughout the central nervous system even when prions were injected directly into the sciatic nerve. These results indicate that a lympho-neural sequence of spread occurs in wild-type mice even upon intranerval inoculation, while overexpression of PrPC leads to substantial predilection of intranerval over lymphoreticular spread. The rate of transport of infectivity in peripheral nerves was ca. 0·7 mm per day, and prion infectivity titres of sciatic nerves were much higher in tga20 than in wild-type mice, suggesting that overexpression of PrPC modulates the capacity for intranerval transport.
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