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1.
Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness. 相似文献
2.
Detection of cytopathic and noncytopathic bovine viral diarrhea virus in cell culture with an immunoperoxidase test 总被引:3,自引:0,他引:3
Bovine viral diarrhea virus (BVDV) antigen was detected in cell culture with an indirect immunoperoxidase (IP) procedure using a specific monoclonal antibody, and an avidin-biotin-peroxidase complex. Cytopathic and noncytopathic strains of the virus showed similar patterns of IP staining until 3 days post-infection. At six days post-infection, intensity of staining decreased in cell cultures infected with noncytopathic virus, but not with cytopathic virus. The IP procedure detected BVDV antigen in cells used to isolate virus from tissues of aborted bovine fetuses and peripheral blood lymphocytes of adult cattle. Immunoperoxidase detected BVDV isolates from 10 of 44 cases of abortion of which seven of these were noncytopathic. Noncytopathic BVDV isolates from the peripheral blood lymphocytes of 7 of 65 animals were identified. 相似文献
3.
Two biotypes of bovine viral diarrhea-mucosal disease virus are present in nature: one that induces cytopathology in infected bovine cells and the other that infects cells without overt cytopathology. Infections with both types of virus yield similar amounts of infectious progeny virus. Field and laboratory isolates of both biotypes of bovine viral diarrhea (BVD) virus were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis of infected cell extracts. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of peculiar polypeptide profiles they induce in the infected cell. The most abundant polypeptide in CB-BVD infected cells is the 80K polypeptide. NCB-BVD virus-infected cells lack the 80K polypeptide and induce a predominant 118K polypeptide. D-[2-3H]Mannose labeling of cells infected with NCB-BVD indicated that at least three polypeptides are N-glycosylated: 75K, 56K-58K, and 48K. In addition the sizes and ratios of the glycoproteins induced by all virus isolates showed a marked variation. We present evidence indicating that there is remarkable heterogeneity among the field viral isolates of BVD and this methodology is of potential value for molecular epidemiology studies. 相似文献
4.
Bovine viral diarrhea virus is an important animal pathogen. The cytopathic and noncytopathic biotypes of the virus are associated with distinct pathologic entities. A striking difference between the two biotypes is viral RNA accumulation in infected cells. Viral dsRNA is thought to activate protein kinase PKR; an important mediator of innate immunity. In this study, we investigated PKR activation and its consequences in BVDV-infected cells. Infection with cp BVDV was found to induce PKR activation, eIF2alpha phosphorylation, translation inhibition and NF-kappaB activation. In contrast, PKR activity and eIF2alpha phosphorylation were not induced during infection with the ncp BVDV. In addition, cells infected with ncp BVDV showed no PKR phosphorylation in response to infection with the unrelated poliovirus whereas uninfected ncp BVDV cells when infected with poliovirus showed high levels of phosphorylated PKR. Cells infected with ncp BVDV failed to respond to synthetic dsRNA (poly I:C) treatment with NF-kappaB activation. However, the NF-kappaB response to bacterial lipopolysaccarides (LPS) was normal in these cells, suggesting a specific suppression of antiviral response signaling in ncp BVDV infected cells. These results indicate that ncp BVDV has evolved specific mechanisms to prevent activation of PKR and its antiviral effectors, most likely to facilitate the establishment and maintenance of persistent infection. 相似文献
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Neutralizing antibodies to type 1 and 2 bovine viral diarrhea viruses: detection by inhibition of viral cytopathology and infectivity by immunoperoxidase assay. 总被引:4,自引:0,他引:4
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R W Fulton J T Saliki L J Burge J M d'Offay S R Bolin R K Maes J C Baker M L Frey 《Clinical and Vaccine Immunology : CVI》1997,4(3):380-383
Neutralizing antibodies to type 1 and 2 bovine viral diarrhea virus (BVDV) strains were measured by a microtiter virus neutralization test (MVNT) in cell culture. Antibodies (neutralizing) were detected by inhibition of viral infectivity, by the absence of viral cytopathology for cytopathic strains, or by immunoperoxidase staining for noncytopathic strains. The immunoperoxidase-stained monolayers could be detected without the aid of light microscopy. Twenty BVDV strains were used as challenge viruses in the in vitro MVNT, including 14 type 1 and 6 type 2 strains. Representative noncytopathic and cytopathic strains of both types were used. Positive control serum samples available for diagnostic testing contained both type 1 and type 2 BVDV antibodies. There did not appear to be major differences in antibody titers among the respective type strains, regardless of biotype (cytopathic or noncytopathic). In a study with sera from calves receiving a modified live virus or inactivated BVDV vaccine, the calves receiving type 1 strains responded with higher antibody titers to type 1 strains than to type 2 strains. 相似文献
7.
R K Akkina 《Virus research》1991,19(1):67-81
The genome of pestivirus bovine viral diarrhea virus (BVDV) contains a single 11,964 nt long open reading frame (ORF) that is capable of encoding a 449 kDa putative polyprotein. Although previous studies have described many virus-coded polypeptides that are believed to arise by post- and/or co-translational proteolytic processing in infected cells, there are two separate regions in the ORF for which no polypeptide products could be identified. In the present study using site specific antisera, we identified two new large proteins of Mr 175 and 172 kDa which encompass two previously described smaller precursor proteins, p125 and p133, respectively. These two large precursor proteins together with two other previously described proteins p22 and gp118 (Mr approx. 84 kDa; unglycosylated) account for the predicted Mr of the putative 449 kDa polyprotein precursor. Furthermore, we discovered three additional polypeptides of Mr 168, 96 and 72 kDa encoded by the last third of the genome. These proteins may arise by an alternative cleavage pathway of the precursor protein p172. A modified and updated map of BVDV ORF incorporating the above findings is presented. 相似文献
8.
Two biotypes of bovine viral diarrhea viruses (BVDV), cytopathic (cp) and noncytopathic (ncp), are recognized based on their activity in cultured epithelial cells. Biotype does not correlate to virulence in acute infections as BVDV strains associated with severe acute BVD outbreaks are all noncytopathic based on their growth characteristics in cultured epithelial cells. Previous studies have shown that acute infections with highly virulent BVDV result in depletion of cells in lymphoid tissues. In this study, flow cytometry demonstrated that infection with a highly virulent BVDV strain was associated with a pronounced reduction in circulating white blood cells (WBC) and increased numbers of apoptotic and necrotic circulating WBC in vivo. Infection with low virulence BVDV did not result in a significant increase in death of circulating WBC. Thus, there appeared to be a correlation between depletion of circulating WBC and virulence. To study the interaction of BVDV strains with lymphoid cells in the laboratory, we developed an in vitro model that used a bovine lymphoid cell line (BL-3 cells). Using this model, it was found that while BVDV strains are segregated into two biotypes based on their activity in cultured epithelial cells, they may be segregated into three biotypes based on their activity in cultured lymphoid cells. These three biotypes are noncytopathogenic (no obvious effects on the viability of either cultured epithelial or lymphoid cells), cytopathogenic (cytopathic effect and cell death in both cultured epithelial and lymphoid cells within 48 h of infection) and lymphocytopathogenic (no effect on cultured epithelial cells, however, cell death in cultured lymphoid cells is observed within 5 days of infection). The proposed lymphocytopathic biotype correlates with high virulence in acute infections in vivo. Cell death caused by the lymphocytopathogenic biotype was not associated with changes typically seen with cytopathic viruses grown in cultured epithelial cells (e.g. changes in processing of the NS2/3 protein observed within 24h post infection, crenation and breakdown of cell integrity within the first 48 h post infection). These data suggest that the cytopathic effect induced in cultured lymphoid cells by a ncp highly virulent BVDV strain may occur by a different mechanism than the cytopathic effect induced by cp BVDV strains. 相似文献
9.
Summary This study examined synthetic oligonucleotide probes as potential diagnostic tools for bovine viral diarrhea virus (BVDV). Six 20-base sequences from across the genome were selected by homology analysis of the published genomic sequences of the NADL and Osloss isolates of BVDV. RNA was extracted from 22 BVDV isolates propagated in bovine turbinate (BT) cells, blotted, and probed with32P end-labeled oligonucleotides. The stringency conditions used were such that more than a single base mismatch would result in no hybridization. The probe originating nearest the 5 end of the viral RNA, ND001, detected 86% of the viral isolates while the other probes detected from 19% to 57%. Both cytopathic and noncytopathic isolates were detected by these synthetic probes. A cocktail of these probes were used to specifically detect BVDV RNA extracted directly from tissues of cattle either persistently or acutely infected. 相似文献
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Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus 总被引:11,自引:0,他引:11
Summary Variation of the intracellular polypeptides induced in calf testis cells by 5 cloned isolates of bovine virus diarrhoea virus (BVDV) was examined. Three of the isolates were cytopathic (NADL, C 2415 and Pe 515 c) and two were non-cytopathic (C 1226 and Pe 515 nc) in these cells. The isolates Pe 515 c and Pe 515 nc were both isolated from an animal with clinical signs of mucosal disease. In cells infected with NADL, 8 virus specific proteins (vp 1 to vp 8) with molecular weights ranging from 120,000 (vp 1) to 23,000 (vp 8) were detected. Isolates C 2415 and Pe 515 c gave a similar array of polypeptides to NADL, but the 3 cytopathic isolates could be distinguished by the variation in the molecular weights of some of the proteins. The non-cytopathic isolates could also be distinguished from each other by this type of molecular variation; however, one feature that characterised these strains, when compared to the cytopathic isolates, was the absence of vp 2. Comparison of the polypeptides induced by Pe 515 c and Pe 515 nc showed that apart from the lack of vp 2 in the Pe 515 nc virus profile, the molecular weights of the other viral proteins were similar. This supports serological evidence that for mucosal disease to occur the pair of cytopathic and non-cytopathic viruses must be closely related. Four of the polypeptides induced by Pe 515 c were shown to be glycoproteins.With 5 Figures 相似文献
12.
Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea viruses type 2 总被引:1,自引:0,他引:1
Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype. 相似文献
13.
Electron microscopic studies of bovine viral diarrhea virus in tissues of diseased calves and in cell cultures 总被引:8,自引:0,他引:8
Summary Pathomorfological studies by electron microscopy (EM) were carried out on the intestines and lymphoid tissues, the buffy coat cells and cultured lymphocytes from calves suffering from mucosal disease (MD). This led to the detection of particles, 45–55 nm in diameter, within characteristic vesicular structures. As these findings coincided with the isolation of bovine viral diarrhea virus (BVDV) from the same tissues and demonstration of BVDV-antigen by immunocytochemical techniques in corresponding samples, the particles were tentatively identified as the BVDV.A detailed study ofin vitro infected bovine cell cultures corroborated this supposition and contributed to a conjectural evaluation of the viral morphogenesis. It revealed a difference from the morphogenesis of most other togaviruses, as the presumed virions were assembled within smooth-membraned vesicles, formed during the infection. Thus, in the material examined, a budding process was not involved in the development of BVDV.With 11 Figures 相似文献
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Philippe Mtzener Ioannis Magkouras Till Rümenapf Ernst Peterhans Matthias Schweizer 《Virus research》2009,140(1-2):15-23
Interferon (IFN) type-I is of utmost importance in the innate antiviral defence of eukaryotic cells. The cells express intra- and extracellular receptors that monitor their surroundings for the presence of viral genomes. Bovine viral diarrhoea virus (BVDV), a Pestivirus of the family Flaviviridae, is able to prevent IFN synthesis induced by poly(IC), a synthetic dsRNA. The evasion of innate immunity might be a decisive ability of BVDV to establish persistent infection in its host. We report that ds- as well as ssRNA fragments of viral origin are able to trigger IFN synthesis, and that the viral envelope glycoprotein Erns, that is also secreted from infected cells, is able to inhibit IFN expression induced by these extracellular viral RNAs. The RNase activity of Erns is required for this inhibition, and Erns degrades ds- and ssRNA at neutral pH. In addition, cells infected with a cytopathogenic strain of BVDV contain more dsRNA than cells infected with the homologous non-cytopathogenic strain, and the intracellular viral RNA was able to excite the IFN system in a 5′-triphosphate-, i.e. RIG-I-, independent manner. Functionally, Erns might represent a decoy receptor that binds and enzymatically degrades viral RNA that otherwise might activate the IFN defence by binding to Toll-like receptors of uninfected cells. Thus, the pestiviral RNase efficiently manipulates the host's self–nonself discrimination to successfully establish and maintain persistence and immunotolerance. 相似文献
16.
Bovine viral diarrhea virus induced apoptosis correlates with increased intracellular viral RNA accumulation 总被引:7,自引:0,他引:7
Non-cytopathic (NCP) and cytopathic (CP) parent-daughter pairs are often isolated from cattle with bovine viral diarrhea virus (BVDV) induced mucosal disease. Alignment of these pair genomes revealed that genetic changes in CP BVDV involve the NS2-3 coding region and correlate with expression of NS3. However, additional mutations are present elsewhere in the genomes of these natural pairs, precluding unambiguous mapping of this function to the NS2-3 region. To evaluate this phenomenon in identical genetic backgrounds, we have constructed an NCP isogenic pair of the NADL by deletion of the cIns from NS2 region. The levels of viral protein synthesis in infected cells revealed no marked difference between the CP and the isogenic NCP BVDV mutant. In contrast, RNA accumulation in cells infected with CP virus was up to 25 times higher than that in cells infected with NCP BVDV. No significant difference in growth kinetics and viral yields were observed between the CP BVDV and the isogenic NCP pair. Analyses of additional NCP/CP parent-daughter field BVDV isolates revealed a similar pattern of macromolecular synthesis, suggesting the generality of this phenomenon. These results implicate increased levels of RNA accumulation in CP BVDV infected cells, along with the production of NS3 as potential contributors to viral cytopathogenicity. 相似文献
17.
Friesian calves were infected with one of three isolates of bovine viral diarrhoea virus (BVDV) and used to establish parameters for an in vitro model of BVDV-reactive T-cell responses in cattle. The study assessed virus clearance, seroconversion, maturation of lymphoproliferative responses (both during and following disease resolution) and the antigen-specificity of CD4(+) T cells from recovered animals. Seroconversion and virus-specific lymphoproliferation were not detected until viraemia had resolved. Interestingly, lymphoproliferation was detected earlier in the animals infected with cytopathic viruses than in those infected with noncytopathic virus despite broadly similar rates of virus clearance and seroconversion for both biotypes. CD4(+) and CD8(+) T cells were induced to proliferate by virus-infected stimulator cells whereas only CD4(+) T cells responded to non-infectious antigens. Lymphoproliferation was strain cross-reactive and MHC-restricted. Induction of T-cell proliferation by recombinant proteins identified the major envelope proteins E(rns) and E2 and the nonstructural (NS) 2-3 protein as T-cell determinants. In addition, the capsid (C) and/or the amino-terminal proteinase, N(pro) were identified as T-cell determinants from the responses of short-term T-cell lines. Thus, in this model, the CD4(+) T-cell repertoire induce by acute BVDV infection includes at least the major envelope proteins, NS2-3, and capsid and/or N(pro). 相似文献
18.
Infection of bovine cells with bovine viral diarrhoea virus (BVDV) can be blocked by the monoclonal antibody (mab) BVD/CA 26, which is directed against a cellular membrane protein. To characterize this molecule, it was isolated and purified by column chromatography. It was found to be an acidic, glycosylated membrane protein consisting of two polypeptide chains of about 28 and 56 kDa. Under non-reducing conditions the chains formed multimers of about 200 kDa. In an actin binding assay the 56 kDa polypeptide chain bound to F-actin as judged by co-sedimentation with actin filaments. Since the target molecule of BVD/CA 26 is localized on the surface of living cells and additionally binds to F-actin, a possible biological function may be to connect the cortical actin filaments with the cellular plasma membrane. The blocking effect of BVD/CA 26 indicates that this cellular plasma membrane protein is involved in the endocytic pathway of BVDV particles. 相似文献
19.
When bovine embryonic kidneys collected at the Gorgie Abattoir, Edinburgh were examined for evidence of infection with bovine viral diarrhoea virus (BVDV), 11 out of 26 kidneys were found to be positive. A technique that detected the presence of inadvertent BVDV in cell cultures consisted of processing and digesting the kidneys to produce cell suspensions, adding dimethyl sulphoxide and dispersing the suspensions in small aliquots that were stored frozen at - 114 degrees C. One aliquot was cultured and screened for BVDV by indirect immuno-fluorescence and interference tests. Bovine embryonic kidney cells so processed retained their viability and virus susceptibility for 15 to 18 months. Selected stocks of "clean" cells only are then used for vaccine production or diagnostic purposes. The cytopathic NADL strain of BVDV multiplied in naturally infected cell cultures but the titres attained were significantly lower than in "clean" cell cultures. 相似文献
20.
M. A. Richardson R. W. Boulton R. S. Raghow Lynn Dalgarno 《Archives of virology》1980,63(3-4):263-274
Summary Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses.InAedes albopictus cells, RRV reached peak titres at 34–48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection.During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre.In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9–11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.With 6 Figures 相似文献