RESUMO
Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.
Assuntos
Chaperonas Moleculares , Infecções por Pestivirus , Pestivirus , Suínos , Replicação Viral , Animais , Linhagem Celular , Coenzimas , Genoma Viral/genética , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/genética , Pestivirus/classificação , Pestivirus/enzimologia , Pestivirus/crescimento & desenvolvimento , Infecções por Pestivirus/veterinária , RNA Viral/genética , Suínos/virologia , Doenças dos Suínos/virologia , Proteases Virais/metabolismo , Replicação Viral/genéticaRESUMO
Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus A through Pestivirus K. Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus L through Pestivirus S.
Assuntos
Flaviviridae/classificação , Infecções por Pestivirus/veterinária , Pestivirus/classificação , Filogenia , Animais , Flaviviridae/genética , Pestivirus/genética , Infecções por Pestivirus/virologia , Ruminantes/virologia , Suínos/virologia , Proteínas Virais/genéticaRESUMO
Atypical porcine pestivirus (APPV), currently classified as pestivirus K, causes congenital tremor (CT) type A-II in piglets. Eighteen APPV strains were identified from 2297 South Korean wild boars captured in 2019. Phylogenetic analysis of the structural protein E2 and nonstructural proteins NS3 and Npro classified the APPV viruses, including reference strains, into Clades I, II and III. Clade I was divided into four subclades; however, the strains belonging to the four subclades differed slightly, depending on the tree analysis, the NS3, E2, and Npro genes. The maximum-likelihood method was assigned to South Korean wild boar APPV strains to various subclades within the three trees: subclades I.1 and I.2 in the E2 tree, subclade I.1 in the Npro tree, and subclades I.1 and I.4 in the NS3 ML tree. In conclusion, APPV among South Korean wild boars belonging to Clade I may be circulating at a higher level than among the South Korean domestic pig populations.
Assuntos
Genoma Viral/genética , Infecções por Pestivirus/epidemiologia , Pestivirus/classificação , Pestivirus/genética , RNA Viral/genética , Animais , Endopeptidases/genética , Variação Genética/genética , Geografia , Pestivirus/isolamento & purificação , Infecções por Pestivirus/veterinária , Filogenia , República da Coreia/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Tremor/virologia , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Natural Pestivirus H infections in cattle have been reported worldwide; however, only a few cases of Pestivirus H have been described in non-bovine ruminants such as goats. A new Pestivirus H HN1507 strain was isolated from an infected goat in 2015 and the genome sequence was determined. The full-length genome sequence was 12,556 nucleotides. Phylogenetic analysis, based on the complete genome and Npro fragments, revealed that the isolate belonged to Pestivirus H and was closely related to strains from Italy. Two unique amino acid substitutions were found in the C-terminal of the E2 protein. To the best of our knowledge, this is first report determining the complete genome of a Pestivirus H strain from goat.
Assuntos
Doenças das Cabras/virologia , Infecções por Pestivirus/veterinária , Pestivirus/classificação , Pestivirus/isolamento & purificação , Animais , China , Análise por Conglomerados , Genoma Viral , Genótipo , Cabras , Mutação de Sentido Incorreto , Pestivirus/genética , Infecções por Pestivirus/virologia , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Proteínas do Envelope Viral/genética , Sequenciamento Completo do GenomaRESUMO
Pestiviruses are highly variable RNA viruses belonging to the continuously growing family Flaviviridae. A genetically very distinct pestivirus was recently discovered in the USA, designated atypical porcine pestivirus (APPV). Here, a screening of 369 sera from apparently healthy adult pigs demonstrated the existence of APPV in Germany with an estimated individual prevalence of 2.4% and ~10% at farm level. Additionally, APPV genomes were detected in newborn piglets affected by congenital tremor (CT), but genomes were absent in unaffected piglets. High loads of genomes were identified in glandular epithelial cells, follicular centers of lymphoid organs, the inner granular cell layer of the cerebellum, as well as in the trigeminal and spinal ganglia. Retrospective analysis of cerebellum samples from 2007 demonstrated that APPV can be found in piglets with CT of unsolved aetiology. Determination of the first European APPV complete polyprotein coding sequence revealed 88.2% nucleotide identity to the APPV sequence from the USA. APPV sequences derived from different regions in Germany demonstrated to be highly variable. Taken together, the results of this study strongly suggest that the presence of APPV genomes in newborn piglets correlates with CT, while no association with clinical disease could be observed in viremic adult pigs.
Assuntos
Infecções por Pestivirus/diagnóstico , Pestivirus/isolamento & purificação , Doenças dos Suínos/virologia , Tremor/congênito , Animais , Animais Recém-Nascidos , Autopsia/veterinária , Cerebelo/virologia , Alemanha/epidemiologia , Pestivirus/classificação , Pestivirus/genética , Infecções por Pestivirus/epidemiologia , Filogenia , Prevalência , Suínos , Doenças dos Suínos/epidemiologia , Tremor/virologiaRESUMO
Like other members from the Pestivirus genus, 'HoBi'-like pestiviruses cause economic losses for cattle producers due to both acute and persistent infections. The present study analyzed for the first time PI animals derived from a controlled infection with two different 'HoBi'-like strains where the animals were maintained under conditions where superinfection by other pestiviruses could be excluded. The sequence of the region coding for viral glycoproteins E1/E2 of variants within the swarms of viruses present in the PI calves and two viral inoculums used to generate them were compared. Differences in genetic composition of the viral swarms were observed suggesting that host factors can play a role in genetic variations among PIs. Moreover, PIs generated with the same inoculum showed amino acid substitutions in similar sites of the polyprotein, even in serum from PIs with different quasispecies composition, reinforcing that some specific sites in E2 are important for host adaptation.
Assuntos
Infecções por Pestivirus/virologia , Pestivirus/genética , Filogenia , Proteínas do Envelope Viral/genética , Adaptação Fisiológica , Animais , Bovinos , Clonagem Molecular , Células Epiteliais/patologia , Células Epiteliais/virologia , Expressão Gênica , Pestivirus/classificação , Pestivirus/isolamento & purificação , Cultura Primária de Células , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Conchas Nasais/patologia , Conchas Nasais/virologiaRESUMO
Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276âbp contig encoding a predicted 3635âaa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.
Assuntos
Infecções por Pestivirus/veterinária , Pestivirus/classificação , Pestivirus/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/sangue , Análise por Conglomerados , Reações Cruzadas , Dados de Sequência Molecular , Pestivirus/genética , Infecções por Pestivirus/virologia , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Soro/virologia , Suínos , Estados UnidosRESUMO
The genus Pestivirus comprises four established species: Bovine viral diarrhoea viruses 1 (BVDV-1) and 2 (BVDV-2), Border disease virus (BDV), and Classical swine fever virus (CSFV); and a tentative species, Pestivirus of giraffe. Additional pestiviruses have been identified and suggested for recognition as novel subgroups/species. To achieve a reliable phylogeny as the basis for classification of pestiviruses, a molecular dataset of 56 pestiviruses and 2089 characters, comprising the 5'UTR, complete N(pro) and E2 gene regions was analysed by Maximum likelihood and Bayesian approach. An identical, robust tree topology was inferred, where seven well-supported monophyletic clades and two highly divergent lineages were identified. Dating most recent common ancestor was estimated for major pestivirus lineages and their evolutionary histories were revealed. Accordingly, a new proposal is presented for the classification of pestiviruses into nine species: BVDV-1, BVDV-2, BVDV-3 (atypical bovine pestiviruses), Pestivirus of giraffe, CSFV, BDV, Tunisian sheep virus (TSV; previously termed "Tunisian isolates"), Antelope and Bungowannah.
Assuntos
Evolução Molecular , Pestivirus/classificação , Filogenia , Genoma Viral , Pestivirus/genética , Proteínas do Envelope Viral/genéticaRESUMO
A unique pestivirus, isolated from a pronghorn antelope (Antilocopra americana), was characterized. Serum neutralization studies suggested that this virus was antigenically related to pestiviruses. Genomic characteristics, unique to pestiviruses, indicated that this virus belongs to the Pestivirus genus. These characteristics included the organization of the 5' untranslated region (5'-UTR), the presence and length of a viral Npro coding region, conservation of cysteine residues in Npro, conservation of predicted amino acid sequences flanking the cleavage sites between viral polypeptides Npro and C and between C and Erns and conservation of predicted hydrophobicity plots of Npro protein. While this data indicated the virus belongs to the Pestivirus genus, phylogenetic analysis in 5'-UTR, Npro and E2 regions suggested that it is the most divergent of the pestiviruses identified to date. This conclusion was also supported by the amino acid identity in coding regions. The corresponding values were much lower for the comparison of pronghorn pestivirus to other pestivirus genotypes than only between previous recognized genotypes. These results suggest the virus isolated from pronghorn antelope represents a new pestivirus genotype. It also represents the only pestivirus genotype first isolated from New World wildlife.
Assuntos
Antílopes/virologia , Infecções por Pestivirus/veterinária , Pestivirus/genética , Pestivirus/isolamento & purificação , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Dados de Sequência Molecular , Testes de Neutralização , Pestivirus/classificação , Infecções por Pestivirus/virologia , Filogenia , Processamento de Proteína Pós-Traducional , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Currently, the genus Pestivirus comprises the four approved species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) and one tentative fifth species represented by a single strain (H138) isolated from a giraffe in Kenya more than 30 years ago. To further address the issue of heterogeneity of pestiviruses we have determined the entire N(pro) and E2 coding sequences for several new pestivirus isolates. Interestingly, phylogenetic analysis revealed that one pestivirus isolated in the 1990s in Africa is closely related to strain H138. Moreover, several novel pestiviruses isolated from sheep group together with the previously described strain V60 (Reindeer-1) isolated from a reindeer, whereas one ovine pestivirus strain (Gifhorn) significantly differs from all previously described pestiviruses, including BDV. We propose to term these mainly sheep-derived pestiviruses BDV-2 (V60-like isolates) and BDV-3 (Gifhorn); consequently, the "classical" BDV isolates should be termed BDV-1. As an additional criterion for segregation of pestiviruses, the antigenic relatedness of pestivirus isolates covering all observed major genotypes was studied by cross-neutralization assays. Analysis of the antigenic similarities indicated the presence of seven major antigenic groups corresponding to BVDV-1, BVDV-2, CSFV, BDV-1, BDV-2, BDV-3, and "giraffe". Taking into account the host origin, the lack of differences concerning the course of disease, and the results of our genetic and antigenic analyses, we suggest that BDV-1, BDV-2, and BDV-3 should be considered as major genotypes within the species BDV.
Assuntos
Pestivirus/classificação , Ovinos/virologia , África , Animais , Variação Antigênica , Antígenos Virais/imunologia , Bovinos , Reações Cruzadas , Endopeptidases/genética , Genótipo , Alemanha , Dados de Sequência Molecular , Testes de Neutralização , Pestivirus/genética , Pestivirus/imunologia , Filogenia , Especificidade da Espécie , Reino Unido , Proteínas do Envelope Viral/genéticaRESUMO
The complete Npro coding sequences were determined for 16 pestiviruses isolated from cattle, pig, and several wild ruminant species including reindeer, bison, deer, and bongo. Phylogenetic analysis enabled the segregation of pestiviruses into the established species bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV). For BVDV-1 five distinct subgroups were identified, while BVDV-2, BDV, and CSFV were each subdivided into two subgroups. The virus isolates from bongo and deer as well as one porcine virus isolate belong to BVDV-1. Interestingly, the isolates from reindeer and bison are distinct from the established pestivirus species. The Npro sequences from these two viruses are more similar to BDV than to the other pestivirus species. Calculation of the pairwise evolutionary distances allowed a clear separation of the categories species, subgroup, and isolate only when the reindeer/bison viruses were considered as members of an additional pestivirus species. Furthermore, the entire E2 coding sequences of a representative set of virus isolates covering all recognized species and subgroups were studied. Segregation of pestiviruses based on the E2 region was identical with that obtained with the N(pro) sequences.
Assuntos
Variação Genética/genética , Pestivirus/classificação , Pestivirus/genética , Sequência de Aminoácidos , Animais , Bison , Bovinos , Cervos , Glicoproteínas/análise , Glicoproteínas/genética , Dados de Sequência Molecular , Pestivirus/isolamento & purificação , Infecções por Pestivirus/classificação , Infecções por Pestivirus/diagnóstico , Filogenia , Rena , Homologia de Sequência de Aminoácidos , Suínos , Proteínas do Envelope Viral/genéticaRESUMO
A monoclonal antibody (mAb)-based immunoperoxidase monolayer assay (IPMA) for detection of bovine viral diarrhea virus (BVDV) was developed and compared with an existing bovine polyclonal antibody (pAb)-based IPMA. A pool of 5 mAbs, 4 mAbs produced to a type 1 BVDV and 1 mAb produced to a type 2 BVDV, was utilized in the mAb-IPMA. The mAbs were chosen for inclusion in the pool because of their broad cross-reactivities with type 1 and/or type 2 BVDV, their apparent avidities for antigen, their reactivity to different BVDV proteins, and their lack of competition for binding sites or their binding to unusual BVDV isolates. The mAb-IPMA outperformed the pAb-IPMA in staining, ease of reading test results, and relative sensitivity with a panel of known BVDV positive and negative sera. The relative sensitivities of the mAb-IPMA and pAb-IPMA were 100% and 93.5%, respectively, for 62 positive samples including several that were known to contain type 2 BVDV. With retesting, the pAb-IPMA gave a similar level of sensitivity as that of the mAb-IPMA. Both tests gave a specificity of 100% for 40 negative serum samples obtained from a BVDV-free herd.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Pestivirus/classificação , Pestivirus/isolamento & purificação , Animais , Anticorpos , Anticorpos Monoclonais , Bovinos , Reações Cruzadas , Técnicas Imunoenzimáticas/veterinária , Sensibilidade e EspecificidadeRESUMO
Conventionally, the genus Pestivirus of the family Flaviviridae has been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). To date, BDV and BVDV have been isolated from different species, whereas CSFV seems to be restricted to swine. Pestiviruses are structurally and antigenically closely related. Envelope glycoprotein E2 is the most immunogenic and most variable protein of pestiviruses. We cloned E2 genes of many different pestivirus strains, including those from a deer and a giraffe. The E2 genes were transiently expressed, characterized with monoclonal antibodies, sequenced, and compared. Based on these data, we can delineate six major groups within the Pestivirus genus. Four groups correspond to defined genotypes, whereas the two other groups could be new genotypes within the Pestivirus genus. One group comprises CSFV strains isolated from swine. A second group consists of BDV strains Moredun, L83, and X818, which have been isolated from sheep, and strain F from swine. A third group contains strain BD78 from sheep, strain 5250 from swine, and strain 178003 from cattle. On the basis of E2, these viruses are very similar to BVDV strains associated with acute severe outbreaks of bovine viral diarrhea, so-called type 2 BVDV. The fourth group consists of BVDV strains originating predominantly from cattle. This BVDV group can be divided into two subtypes or subgroups BVDV Ia and Ib: BVDV Ia contains viruses from the United States, such as like NADL and Oregon, and some others, such as 150022 and 1138 from Europe. Subgroup BVDV Ib contains strain Osloss and several Dutch isolates. The fifth and sixth "groups" could be proposed as two new genotypes and contain strains Deer and Giraffe, respectively.
Assuntos
Pestivirus/classificação , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Bovinos , Dados de Sequência Molecular , Pestivirus/genética , Alinhamento de Sequência , Análise de SequênciaRESUMO
The genus Pestivirus is composed of hog cholera virus (HCV) [also known as classical swine fever virus (CSFV)], bovine viral diarrhea virus (BVDV), and border disease virus (BDV). Complete sequences have been published for HCV (or CSFV) and the two genotypes of BVDV (BVDV1 and BVDV2). In this study the complete sequence of the border disease virus (BDV), BD31, was determined. BD31 was isolated from a lamb with hairy shaker syndrome and is the BDV type virus offered by ATCC (ATCC VR-996). The genome was 12268 nucleotides long and had a single large open reading frame (ORF) beginning at nucleotide 357 and ending at nucleotide 12045. The sequence identity of the predicted amino acid sequence of BD31 and other published pestivirus sequences varied from 71% to 78%. Phylogenetic analysis of available complete genomic sequences segregated pestiviruses into two branches. One branch contained BD31 and HCV (or CSFV) isolates while the other branch contained BVDV1 and BVDV2 isolates. Pestiviruses from the same branch were similar in the length of the 5' and 3' untranslated regions (UTR). When complete genomic sequences were compared among BD31, HCV (or CSFV), BVDV1 and BVDV2, the highest sequence identity was observed in the 5' UTR. Within the ORF, the highest sequence identity was observed in the genomic region coding for the nonstructural viral polypeptide p80.
Assuntos
Vírus da Doença da Fronteira/química , Vírus da Doença da Fronteira/genética , Genoma Viral , Pestivirus/química , Pestivirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Vírus da Doença da Fronteira/classificação , Dados de Sequência Molecular , Pestivirus/classificação , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , OvinosRESUMO
Antigenic and genetic analyses were performed in order to establish relationships between the noncytopathogenic (ncp) and the cytopathogenic (cp) bovine viral diarrhoea viruses (BVDV) involved in the induction of a case of experimentally induced "late-onset" mucosal disease (MD) symptoms. The persistent ncpBVDV, the cpBVDV used for superinfection (strain TGAC) and the virus isolates from faeces (cpX) were examined using an immunoplaque test (IPT) to distinguish between cp and ncp virus populations. The cp populations were cloned by plaque purification and found to be free of ncpBVDV when using the IPT. The cpBVDV clones and the persistent ncpBVDV were analysed in an enzyme immunoassay on heat-fixed infected cells (IM-EIA) and in a neutralization test using a panel of 27 monoclonal antibodies against the E0 (gp48) and E2 (gp53) viral glycoproteins. It was found that strain TGAC contained two antigenically distinct subpopulations of cpBVDV (TGAC-B1 and TGAC-B2). The endogenous ncpBVDV and the cpX clones had the same reactivity pattern in both tests. In addition, p80 gene duplications in the genomes of the cpBVDV clones were analysed using the polymerase chain reaction and subsequent restriction enzyme analysis of the amplicons. The clones analysed from TGAC-B1 and those from cpX had gene duplications of identical sizes showing the same restriction enzyme patterns. Our results suggest that the cpBVDV which finally lead to "late-onset" MD arose by recombination and/or by mutations of the cpBVDV used for superinfection.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/fisiopatologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Pestivirus/isolamento & purificação , Animais , Anticorpos Monoclonais , Antígenos Virais/análise , Sequência de Bases , Bovinos , Células Cultivadas , Primers do DNA , DNA Viral/análise , Epitopos/análise , Técnicas Imunoenzimáticas , Rim , Dados de Sequência Molecular , Testes de Neutralização , Pestivirus/classificação , Pestivirus/patogenicidade , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Ensaio de Placa ViralRESUMO
Sixty-six pestiviruses from ruminant and porcine hosts were analysed with a panel of 76 monoclonal antibodies raised against 9 different viruses. Reactivity was used to construct epitope similarity maps for all of the viruses. Four principal virus subgroups were demonstrated. One subgroup equated to classical swine fever virus (CSFV) and included most porcine pestiviruses but none from ruminants. A second subgroup contained mainly viruses of bovine origin, including reference bovine viral diarrhoea viruses (BVDV) such as NADL; however viruses from pigs and sheep were also represented. A third subgroup represented by reference strains of border disease virus (BDV) comprised mainly ovine isolates, but also viruses from pigs. The fourth and most recently defined subgroup contained no reference strains of CSFV, BVDV or BDV, but included atypical viruses from cattle, sheep and pigs. The subgrouping scheme was supported by genetic comparisons between representative viruses from the 4 subgroups and by virus neutralisation with polyclonal sera.
Assuntos
Pestivirus/classificação , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Sequência de Bases , Mapeamento de Epitopos/veterinária , Técnicas Imunoenzimáticas/veterinária , Dados de Sequência Molecular , Testes de Neutralização/veterinária , Pestivirus/genética , Pestivirus/imunologia , Filogenia , Ensaio de Radioimunoprecipitação/veterinária , Ruminantes/virologia , Suínos/virologiaRESUMO
Three serologically different pestivirus strains isolated from sheep were selected for molecular analysis. cDNA and deduced amino acid sequences of the genomic regions encoding glycoproteins E1 and E2 were obtained from the three strains. A comparison with amino acid sequences of bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) revealed that one of the three ovine pestivirus strains can be grouped together with BVDV. The other two strains, however, were clearly different from both BVDV and CSFV. Surprisingly, the amino acid sequences from these two viruses were more similar to CSFV than to BVDV. The identity between so-called "true" BDV strains at the amino acid level is about 95% for E1 and 86% for E2 and thus similar to homologies found between CSFV strains. For one "true" BDV strain the genomic region encompassing the nonstructural protein p125 was also cloned and sequenced. The respective comparative analysis led to results which are similar to the ones obtained for the two structural glycoproteins. Taken together the data demonstrate that "true" border disease virus strains represent a separate group within the genus pestivirus.
Assuntos
Vírus da Doença da Fronteira/genética , Genoma Viral , Pestivirus/genética , Ovinos/microbiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Vírus da Doença da Fronteira/classificação , Clonagem Molecular , Glicoproteínas/genética , Dados de Sequência Molecular , Pestivirus/classificação , Testes de Precipitina , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy of gene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.
Assuntos
Pestivirus/classificação , Proteínas Virais/genética , Animais , Capsídeo/análise , Capsídeo/genética , Genoma Viral , Glicoproteínas/análise , Glicoproteínas/genética , Fases de Leitura Aberta , Pestivirus/química , Pestivirus/genética , Proteínas do Core Viral/análise , Proteínas do Core Viral/genética , Proteínas Virais/análiseRESUMO
Ten monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non-cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent Mr 80,000 and 130,000 from cells infected with cytopathic virus and a single polypeptide of apparent Mr 120,000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.
Assuntos
Antígenos Virais/imunologia , Pestivirus/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais , Antígenos Virais/biossíntese , Doença da Fronteira/microbiologia , Células Cultivadas , Epitopos , Imunofluorescência , Immunoblotting , Camundongos , Peso Molecular , Pestivirus/classificação , Proteínas Virais/biossínteseRESUMO
The molecular features of bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus currently classified in the Togaviridae, were examined for characteristics resembling those of the Flaviviridae family. Like flaviviruses, BVDV possesses a single-stranded RNA genome (approx. 4.3 x 10(6) Mr) deficient in a 3' poly(A) tract. This RNA has a single open reading frame spanning the length of the genome in the viral RNA sense (positive polarity), implying an expression strategy involving the processing of a precursor polyprotein. With the exception of several short but significant stretches of identical amino acids within two non-structural proteins, no extended regions of nucleotide or amino acid sequence homology between BVDV and representatives of three serological subgroups of mosquito-borne flaviviruses were noted. However, comparison of the organization of protein-coding domains along the genomes and the hydropathic profiles of amino acid sequences revealed pronounced similarities. It is proposed that Pestivirus, of which BVDV is the prototype member, should no longer be grouped in the Togaviridae family, but rather be considered a genus of non-arthropod-borne viruses within the Flaviviridae.