RESUMO
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.
Assuntos
Glicoproteínas/biossíntese , Pestivirus/metabolismo , Proteínas Virais/biossíntese , Animais , Doença da Fronteira/microbiologia , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/análise , Glicoproteínas/análise , Glicoproteínas/imunologia , Peso Molecular , Mapeamento de Peptídeos , Peptídeos/análise , Pestivirus/análise , Testes de Precipitina , Proteínas Virais/análise , Proteínas Virais/imunologiaRESUMO
Intracellular virus-induced polypeptides from 3 cytopathogenic and 2 non-cytopathogenic bovine viral diarrhea (BVD) virus reference strains were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis, using a specific bovine multivalent antiserum and a neutralizing BVD-virus monoclonal antibody. Electrophoretic patterns of major proteins demonstrate extensive variation between strains. Most notably, a major 80,000 (80K) polypeptide was present in all cytopathogenic strains but absent in both non-cytopathogenic strains. Furthermore, a neutralizing monoclonal antibody produced against the NADL strain immunoprecipitated a 53K glycoprotein indicating that this protein carries an important neutralization epitope that is not present in all strains tested.
Assuntos
Antígenos Virais/análise , Vírus da Diarreia Viral Bovina/análise , Pestivirus/análise , Proteínas Virais/análise , Animais , Especificidade de Anticorpos , Variação Antigênica , Vírus da Diarreia Viral Bovina/imunologia , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Imunofluorescência , Imunoensaio , Testes de Neutralização , Proteínas Virais/imunologiaRESUMO
The presence of virus-specific polypeptides in bovine viral diarrhoea-mucosal disease (BVD) virus-infected bovine cells was studied by radiolabelling in the presence of a hypertonic initiation block (HIB) and by analysis by SDS-PAGE. These experiments were complemented by radioimmunoprecipitations with anti-BVD hyperimmune serum of infected cells labelled under isotonic conditions. A total of 12 polypeptides (Mr 165, 135, 118, 80, 75, 62, 56 to 58, 48, 37, 32, 35 and 19, all X 10(-3)) were identified in infected cells. Time course analysis of the induction of the viral polypeptides indicated that they could be detected as early as 4 h post-infection and their synthesis reached a plateau between 12 and 20 h post-infection. The most abundant polypeptides were the ones that could be detected earliest. HIB was found to be an excellent adjunct to existing techniques in the identification of viral polypeptides. Seven of these polypeptides had not been reported previously. This is the first report of the direct detection of BVD virus-induced polypeptides in infected cells without the aid of immunoprecipitation. The sum of the molecular masses of these polypeptides is greater than the coding capacity of the genome; therefore precursor-product relationships must exist between these polypeptides.
Assuntos
Vírus da Diarreia Viral Bovina/análise , Pestivirus/análise , Proteínas Virais/análise , Animais , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/metabolismo , Vírus da Diarreia Viral Bovina/fisiologia , Eletroforese em Gel de Poliacrilamida , Soluções Hipertônicas , Técnicas Imunológicas , Cinética , Peso Molecular , Proteínas Virais/biossínteseRESUMO
Bovine cell cultures infected with bovine viral diarrhoea-mucosal disease (BVD) virus were radiolabelled with L-[35S]methionine or D-[2-3H]mannose followed by analysis of the labelled polypeptides by radioimmunoprecipitation and polyacrylamide gel electrophoresis in one and two dimensions. Six glycoproteins were detected in infected cells. Two abundant species had Mr of 48K and 56K to 58K while the less abundant species had Mr of 118K, 75K, 65K and 25K. When cells were radiolabelled with L-[35S]methionine in the presence of tunicamycin 56K to 58K migrated with apparent masses of 54K (a minor species) and 48K to 50K (the major molecular species) in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4K to 6K reduction in the apparent molecular mass of 56K to 58K yielding a single 52K digested product, indicating that the heterogeneity of 56K to 58K was due to differences in the oligosaccharide moieties. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a critical role in the infectious cycle of BVD virus.
Assuntos
Vírus da Diarreia Viral Bovina/análise , Glicoproteínas/análise , Pestivirus/análise , Proteínas Virais/análise , Animais , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/metabolismo , Vírus da Diarreia Viral Bovina/fisiologia , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/biossíntese , Glicosídeo Hidrolases , Glicosilação , Técnicas Imunológicas , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular , Tunicamicina/farmacologia , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , Pestivirus/metabolismo , Proteínas Virais/biossíntese , Animais , Bovinos , Células Cultivadas , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina/análise , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/fisiologia , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/biossíntese , Manose/metabolismo , Peso Molecular , Proteínas Virais/análiseRESUMO
Bovine viral diarrhea virus was purified by lectin chromatography. The glycoprotein peplomers were dissociated from the virion by treatment with detergent. By a second lectin gel chromatography the glycoconjugates containing terminal galactose were prepared. In combination with lectin affinity chromatography, ion-exchange chromatography on Mono-Q in the presence of the low-UV-absorbing detergent Berol 172 proved to be a powerful technique both for analytical and preparative applications.
Assuntos
Detergentes , Vírus da Diarreia Viral Bovina/análise , Proteínas de Membrana/isolamento & purificação , Pestivirus/análise , Tensoativos , Proteínas Virais/isolamento & purificação , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Vírus da Diarreia Viral Bovina/isolamento & purificação , Glicoproteínas/isolamento & purificação , Micelas , Vírion/análiseRESUMO
Bovine viral diarrhea virus (BVDV) was concentrated and purified by a combination of ultrafiltration, hydroextraction using polyethylene glycol and affinity chromatography. A lectin from Crotalaria juncea that has an affinity for galactose was used in the affinity chromatography. Virions of BVDV with classic envelopes were observed by electron microscopy. Four major proteins with estimated molecular weights of 75,000, 66,000, 54,000, and 26,000 were identified in sodium dodecyl sulfate--polyacrylamide gel electrophoresis slab gels. The proteins of 75,000 and 54,000 were glycoproteins as shown by staining with dansyl hydrazine.
Assuntos
Vírus da Diarreia Viral Bovina/análise , Pestivirus/análise , Proteínas do Envelope Viral/análise , Vírus da Diarreia Viral Bovina/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Proteínas do Envelope Viral/isolamento & purificação , Vírion/análiseRESUMO
The sedimentation coefficient and buoyant density of hog cholera, bovine viral diarrhea and Border disease viruses, have been compared with those of representative members of the family togaviridae. It appears that the pestivirus genus is a homogeneous group which is not only antigenically but also structurally, unrelated to the other genera of the togavirus family.
Assuntos
Arbovírus/análise , Vírus da Febre Suína Clássica/análise , Vírus da Diarreia Viral Bovina/análise , Vírus de RNA/análise , Arbovírus/classificação , Centrifugação com Gradiente de ConcentraçãoRESUMO
Radiolabelled bovine viral diarrhoea/mucosal disease virus (BVDV) strains NADL and Oregon C24V were purified by different steps. Following immunoprecipitation, electrophoresis in SDS-polyacrylamide gels revealed three BVDV structural polypeptides with molecular weights of 57 (VP1), 44 (VP2), and 34 (VP3) kd. The two larger BVDV polypeptides VP1 and VP2 were found to be glycosylated (gp57, gp44). The data obtained on BVDV structural proteins demonstrate common features with hog cholera virus and indicate a common grouping with the family Togaviridae.
Assuntos
Vírus da Diarreia Viral Bovina/análise , Peptídeos/análise , Vírus de RNA/análise , Proteínas Virais/análise , Arbovírus/classificação , Técnicas de Cultura , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/isolamento & purificação , Glicoproteínas/análise , Peso MolecularRESUMO
Pig rotavirus was purified from faeces. The RNA from this virus was resistant to pancreatic ribonuclease, indicating that it is double-stranded. When electrophoresed on polyacrylamide-agarose gels, pig rotavirus RNA migrated as 9 bands comprised of 11 or 12 RNA segments with a total mol. wt. of approx. 11 X 10(6). Co-electrophoresis experiments revealed that the RNAs from the pig virus and two isolates of the calf rotavirus were indistinguishable.
Assuntos
Diarreia/veterinária , Vírus de RNA/análise , RNA Viral , Doenças dos Suínos/microbiologia , Animais , Diarreia/microbiologia , Vírus da Diarreia Viral Bovina/análise , Fezes/microbiologia , Peso Molecular , RNA Viral/análise , SuínosRESUMO
Two distinct types of rotavirus particle were isolated in caesium chloride density gradients. The higher density particle banded at 1-38 g/ml and measured 55 +/- 0-4 nm in diameter while the less dense particle banded at 1-36 g/ml, measured 66 +/- 0-4 nm in diameter and appeared to possess an extra outer capsid layer. Some forms intermediate between the two types were seen. Since the two particles had a similar but not identical morphology and polypeptide composition, they were considered to be different forms of the same virus. Infectivity was associated with the larger particle which contained one more polypeptide than the smaller particle.
Assuntos
Vírus da Diarreia Viral Bovina , Vírus de RNA , Animais , Bovinos , Vírus da Diarreia Viral Bovina/análise , Vírus da Diarreia Viral Bovina/patogenicidade , Vírus da Diarreia Viral Bovina/ultraestrutura , Fezes/microbiologia , Peptídeos/análise , Vírus de RNA/análise , Proteínas Virais/análiseRESUMO
Polyacrylamide gel electrophoretic analysis of purified preparations of human and calf diarrhea viruses indicated eight polypeptide components, or possibly nine in the case of the calf diarrhea virus. Thermal denaturation and analytical studies of the calf diarrhea virus genome showed it to consist of 11 double-stranded segments of RNA. The placing of the human and calf diarrhea viruses together with other similar viruses into a genus separate from reovirus and orbivirus, but within the family Reoviridae, is discussed.