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RNA extracted from isopycnically banded [3-H]uridine-labeled bovine viral diarrhea virus with sodium dodecyl sulfate was resolved into one major and two minor components by both sedimentation analysis and electrophoresis in polyacrylamide gels. The major RNA component was estimated to have a 38S sedimentation coefficient. The minor RNA components were estimated to have S values of 31 and 24. The approximate colecular weights were calculated to be 3.22 times 10-6 (38S), 2.09 times 10-6 (31S), and 1.22 times 10-6 (24S). A single broad peak of radioactivity, maximum at 24S, was obtained when sedimentation was conducted under conditions of low ionic strength. All three RNA components were found to be susceptible to digestion with RNase. The presence of multiple RNA components in heterogeneous populations of infectious virus is discussed. 相似文献
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Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to program cell-free synthesis, mature viral 80,000- and 115,000-molecular-weight proteins were detected; no precursor to the viral 55,000-molecular-weight glycoprotein was noted. The implications of these results with respect to virus-specific protein synthesis are discussed. 相似文献
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Lai VC Kao CC Ferrari E Park J Uss AS Wright-Minogue J Hong Z Lau JY 《Journal of virology》1999,73(12):10129-10136
Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities. 相似文献
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Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus. 总被引:5,自引:3,他引:2
Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed. 相似文献
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John D. Neill 《Biologicals》2013,41(1):2-7
Bovine viral diarrhea viruses (BVDV) are arguably the most important viral pathogen of ruminants worldwide and can cause severe economic loss. Clinical symptoms of the disease caused by BVDV range from subclinical to severe acute hemorrhagic syndrome, with the severity of disease being strain dependent. These viruses are classified as members of the Pestivirus genus of the Flaviviridae. BVDV are considered primarily a pathogen of cattle but can infect most ruminant species. The virus particle consists of a lipid bilayer membrane surrounding the encapsidated genomic RNA. Inserted in the outer membrane are two virus-encoded glycoproteins that contain the major antigenic determinants of the virus as well as receptor binding and cell fusion functions. A third glycoprotein is weakly associated with the virion, but also possesses unique features that play important roles in suppression of innate immunity. The viral proteins are encoded in a single, large open reading frame. The viral proteins are proteolytically cleaved from the polyprotein by different proteases. The structural proteins are processed by cellular signal peptidases while the processing of the nonstructural proteins is by the viral serine protease. The virus is assembled and matures in the endoplasmic reticulum and golgi bodies of the cell. The virus is released via exocytosis, where viral proteins are not exposed on the surface of the cell. 相似文献
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Laboratory diagnosis of bovine viral diarrhea virus 总被引:1,自引:0,他引:1
Edward J. Dubovi 《Biologicals》2013,41(1):8-13
The control and eventual eradication of bovine viral diarrhea virus (BVDV) have been defined as objectives to reduce the economic losses due to the presence of this virus in the cattle population. These goals could not be envisioned without the significant achievements in the diagnostic procedures employed to detect the infection in its various manifestations. The tests that are currently available are fully capable of supporting the ACVIM consensus statement for the control and eradication of BVDV. In point of fact diagnostic testing is the essential component of any control program. What is now currently lacking is full implementation of the ACVIM recommendations. 相似文献
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Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05). 相似文献
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Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis 总被引:5,自引:0,他引:5
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Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Both noncytopathic (ncp) and cytopathic (cp) biotypes of BVDV can be isolated from persistently infected cattle suffering from the lethal mucosal disease. The cp biotype correlates with the production of the NS3 nonstructural protein, which in the corresponding ncp biotype is present in its uncleaved form, NS23. Previously, we have shown that cp but not ncp BVDV induces the formation of alpha/beta interferons in bovine macrophages. In this study, we demonstrate that ncp BVDV inhibits the induction of apoptosis and the expression of interferon alpha/beta by poly(IC), a synthetic double-stranded RNA (dsRNA). Inhibition was observed only in cells which had been infected with ncp BVDV at least 12 h prior to the addition of dsRNA, which indicates that expression of viral proteins is necessary for the ncp virus to inhibit the effects of poly(IC). Additional experiments using transfected poly(IC) showed that ncp BVDV interfered with the intracellular action of dsRNA rather than with its uptake into the cells. Infected cells were not resistant to induction of apoptosis by actinomycin D or staurosporine, which suggests that ncp BVDV may specifically interfere with signaling through dsRNA. Interference with the innate antiviral host responses may explain the successful establishment of persistent infection by ncp BVDV in fetuses early in their development. 相似文献
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D'Abramo CM Deval J Cameron CE Cellai L Götte M 《The Journal of biological chemistry》2006,281(34):24991-24998
The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme. 相似文献
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Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100- to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor. 相似文献
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Grooms DL 《Theriogenology》2006,66(3):624-628
Bovine viral diarrhea virus and Leptospira spp. are two of the common pathogenic organisms responsible for reproductive losses in cattle worldwide. Both can be come endemic in herds resulting in chronic low-grade reproductive losses or they can be introduced into relatively na?ve herds, resulting in substantial reproductive losses over a short period of time. Both organisms are a differential diagnoses for common reproductive losses that veterinarians investigate, including low conception rates and abortions. 相似文献
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Virus-specific proteins were examined in cultured cells infected with bovine viral diarrhea virus. By using antisera obtained from virus-infected animals, three major virus-specific polypeptides with molecular weights of 115,000 (115K), 80K, and 55K were observed. Minor proteins of 45,000 and 38,000 daltons were also noted. Tryptic peptide mapping indicated that the 115K and the 80K polypeptides were structurally related. The 55K protein was glycosylated and appeared not to be related to the 115K and 80K proteins. Pulse-chase experiments failed to demonstrate any procursor-product relationship among any of these proteins, and all three polypeptides were found in purified virion preparations. The significance of these findings with respect to the replication of bovine viral diarrhea virus is discussed. 相似文献
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Kenny V Brock 《Biologicals》2003,31(2):133-135
Bovine viral diarrhoea virus (BVDV) has a unique capacity to cause persistent infections of foetuses exposed within the first 150 days of gestation. Preventing foetal BVDV infection will aid in improved control. This unique ability gives BVDV a selective advantage allowing continual mutation and antigenic variation within cattle populations. Therefore, BVDV has become widespread and causes economic losses due to respiratory, reproductive and enteric disease. Vaccination (modified-live or killed) can provide some protection from acute disease and the development of persistently infected foetuses. However, vaccination programmes alone cannot control or eliminate BVDV. In naturally exposed and vaccinated herds, BVDV infections are not self-limiting and may persistent over time. This underscores the ability of the BVDV genome to remain fluid and adapt under selective pressures. Factors influencing persistence of BVDV infections in cattle populations include: non-lytic infections; evasion of host immune responses; foetal infections; acute infections; management practices; contaminated biologics; secondary hosts; defective replicated intermediates; antigenic variation; and replication in privileged anatomical sites. 相似文献
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Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is approximately 2.2 microM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 micro M compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 microM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase. 相似文献
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