RESUMO
Bovine viral diarrhea virus (BVDV) infects different cell types including antigen-presenting cells such as macrophages. The infection induces pro-inflammatory cytokines like interleukin 1 beta (IL-1ß), which is necessary to trigger a successful inflammatory response against infections. Several authors have reported differences between IL-1ß gene expression and protein detection in BVDV-infected macrophages. These patterns may be related to inflammasome assembly, which promote the formation of active caspase 1 in order to produce mature IL-1ß molecules. Our goal was to assess BVDV ability to induce the release of IL-ß through a caspase 1 dependent pathway in bovine macrophages. We infected peripheral blood monocyte-derived macrophages using BVDV NADL strain at 0.001, 0.1, 2 and 10 multiplicities of infection (MOI) and we measured IL-1ß at different times 2, 6, 12, 24, 48, 72 h. We found an increase of 1140-2154 pg for a MOI of 10:1 and 2:1 respectively. To inhibit caspase 1, we used either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD) or carbobenzoxy-tyr-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Y-VAD). We found decreased IL-1ß secretion 2154 pg/ml to 854 pg/ml IL-1ß secretion using Y-VAD and we observed decrease from 2154 pg/ml to 22.33 pg/ml with Z-VAD, and this inhibition was followed by diminished viral replication from 2.25 × 107 to 2.1 × 105 CCID50, which suggests that caspase 1-dependent secretion of the IL-1ß active molecule is important for viral replication. This is the first report showing that BVDV infected-bovine macrophages trigger the caspase 1 dependent pathway for IL-1ß activation and that activation increases viral replication.
Assuntos
Caspase 1/metabolismo , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Animais , Bovinos , Citocinas/metabolismo , Regulação da Expressão Gênica , Inflamassomos , Replicação ViralRESUMO
Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.
Assuntos
Bioensaio , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Células Epiteliais/virologia , RNA Viral/genética , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Linhagem Celular , Coinfecção , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 1/metabolismo , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/metabolismo , Cães , Células Epiteliais/patologia , Feminino , Células Madin Darby de Rim Canino , Gravidez , RNA/genética , RNA/metabolismo , Sondas RNA/genética , Sondas RNA/metabolismo , RNA Viral/metabolismo , Tropismo Viral , Replicação ViralRESUMO
Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus within the family Flaviviridae. Based on the 5' untranslated region (UTR) sequence, BVDV-1 can be divided into at least 17 subtypes (1a though 1q). BVDV-1i is an uncommon subtype that has been reported in the United Kingdom and Uruguay. Here, we report the complete genome sequence of the first subtype 1i BVDV-1 (strain ACM/BR/2016) isolated from cattle in southern Brazil. The genome is 12,231 nt in length and contains a single ORF that encodes a polyprotein of 3,896 amino acids, flanked by 5' and 3'UTRs of 325 and 220 nt, respectively. Phylogenetic inferences based on the whole genome, the 5'UTR, and the Npro region showed that strain ACM/BR/2016 is closely related to previously characterized BVDV-1i members. Its 5'UTR shares the highest nucleotide identity (90.5%) with BVDV-1i strains from United Kingdom, and its Npro is most closely related to that of a Uruguayan strain (90.6%). To the best of our knowledge, this is the first BVDV-1i strain from which the whole genome has been completely sequenced and characterized. The complete genome of a BVDV-1i will help future studies on pestivirus evolution and heterogeneity.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Genoma Viral , Regiões 5' não Traduzidas , Animais , Brasil , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/genética , Genômica , Genótipo , Filogenia , RNA Viral/genéticaRESUMO
The SD0803 strain of the bovine viral diarrhea virus (BVDV) was isolated from a piglet in China in 2008 and has been classified as a novel subgenotype of BVDV-1. To describe the molecular features of this novel subgenotype, we sequenced and characterized the complete genome of the SD0803 virus. The genome is 12,271 bp in length and contains 5' and 3' untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a 3,898-amino-acid polypeptide. The full-length genome of the SD0803 strain shares 78.8% to 83.3% identity with those of other BVDV-1 strains, 70.0% to 70.7% identity with those of BVDV-2 strains, and less than 67.6% identity with those of other pestiviruses. The highest level of shared identity was 83.3% between the complete SD0803 genome and that of the ZM-95 strain of BVDV-1. Phylogenetic analysis of the 5' UTR and the coding sequence for the N-terminal protease fragment of the SD0803 polyprotein indicated that the SD0803 virus is a member of the novel subgenotype BVDV-1q, isolates of which have been identified recently in dairy cattle and camels in China.
Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/genética , Genoma Viral , Infecções por Pestivirus/veterinária , RNA Viral/genética , Análise de Sequência de DNA , Suínos/virologia , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , China , Análise por Conglomerados , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Infecções por Pestivirus/virologia , Filogenia , Homologia de SequênciaRESUMO
The aim of this study was to investigate the frequency and diversity of bovine viral diarrhea viruses (BVDV) infecting cattle in Turkey. A total of 1124 bovine blood samples from 19 farms in 4 different Turkish regions were tested by antigen capture ELISA (ACE). BVDV antigen was found in 26 samples from 13 farms. Only 20 of the 26 initial test positive cattle were available for retesting. Of these, 6 of 20 tested positive for BVDV, by ACE and real-time RT-PCR, one month after initial testing. Phylogenetic analysis, based on comparison of the E2 or the 5'UTR coding regions, from 19 of the 26 initial positive samples, indicated that 17 belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of 5'UTR sequences segregated 8 BVDV-1 strains (strains 5, 6, 10, 11, 12, 13, 17, and 19) to the BVDV1f, 1 strain (strain 8) to the BVDV1i and 1 strain (strain 14) to the BVDV1d subgenotypes. One strain (strain 4) did not group with other subgenotypes but was closer to the BVDV1f. The remaining 6 BVDV-1 strains (strains 1, 2, 3, 7, 9, and 18) segregated to a novel subgenotype. The E2 sequence comparison results were similar, with the exception that strain 5 grouped with the novel subgenotype rather than BVDV1f subgenotype. It appears that among the diverse BVDV strains in circulation there may be a subgenotype that is unique to Turkey. This should be considered in the design of diagnostics and vaccines to be used in Turkey.
Assuntos
Bovinos/virologia , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Variação Genética , Proteínas do Envelope Viral/genética , Regiões 5' não Traduzidas , Animais , Antígenos Virais/sangue , Sequência de Bases , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 1/patogenicidade , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/patogenicidade , Ensaio de Imunoadsorção Enzimática , Genótipo , Técnicas de Genotipagem/métodos , Filogenia , Turquia/epidemiologiaRESUMO
Three Indian Bovine viral diarrhea virus 1 (BVDV-1) isolates were analyzed at genetic level in N(pro) (viral autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein, E(rns), and envelope proteins E1 and E2. All these isolates were found to be of b subtype based on the entire 504 nt region of N(pro) and 1119 nt region of E2. However, in comparison with other isolates of this subtype, they were allocated inside the BVDV-1 subtype b cluster to a separate clade with a longer distance. Of six cysteine residues in N(pro) only three were totally conserved in all three isolates. The isolates showed 94.9-99.3% and 92.2-99.0% identities for the entire C-E2 gene region at nucleotide and amino acid levels, respectively. The lowest identity values (88.5-91.7%) were observed for E2 amino acid sequences. The identity of the isolates with Osloss, a reference BVDV-1 subtype b strain, was in the range of 82.1-89.9% for nucleotide and 78.6-89.2% for amino acid sequences in the C-E2 region. The N(pro)/C and E(rns)/E1 cleavage sites were highly conserved. The C/E(rns) and E1/E2 cleavage sites were more conserved from the N-end of E(rns) and the C-end of El, respectively. These findings suggest that some unique mutations have occurred in the described Indian BVDV-1 isolates, though they all belong to the BVDV-1 subtype b.
Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Dados de Sequência Molecular , Mutação , Proteínas Virais/química , Proteínas Estruturais Virais/químicaRESUMO
The aim of this study was to find whether an antigenic drift had occurred in Lower Saxony in the past 40 years. For this, the genetic diversity of bovine viral diarrhea virus (BVDV) isolates mainly from Lower Saxony was estimated by RT-PCR and sequencing of a 420 bp fragment of the E2 glycoprotein gene. Sixty-one field virus isolates collected during routine diagnostics between 1960 and 2000 in Lower Saxony, Northern Germany, were analyzed. Phylogenetic analysis allowed discrimination of genotypes BVDV 1 and 2. Excepting two isolates, which were of BVDV type 2, most of the isolates were classified as BVDV type 1. This group could be further subdivided into four subgroups and one disparate isolate. Independent of the year of isolation and geographical localization, 54 isolates clustered in two subtypes (BVDV subtypes 1b and 1d). Only one isolate was classified as BVDV type 1a, thus being similar to the North American NADL strain, and to the vaccine strain Oregon C24V, which was extensively used for vaccination in Germany. The remaining isolates belonged to new clusters tentatively designated as BVDV subtypes 1g and 1f. To compare the cluster designation with that of other studies, phylogenetic analysis of representatives of each of the subgroups based on the 5' untranslated region (5'UTR) was performed. It grouped the viruses similarly. The results indicate that the BVDV population seems to be relatively stable over 40 years in Lower Saxony.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Vírus da Diarreia Viral Bovina Tipo 2/genética , Regiões 5' não Traduzidas/genética , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Frequência do Gene/genética , Genótipo , Alemanha , Filogenia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Proteínas do Envelope Viral/genéticaRESUMO
Bovine viral diarrhea virus (BVDV) has recently been segregated into two genotypes, namely, BVDV 1 and BVDV 2. Viruses of the BVDV 2 genotype are a cause of hemorrhagic and acute fatal disease in cattle in the US and Canada. In this study, monoclonal antibodies (mAbs) to the newly described BVDV 2 were produced after immunization with virus or a combination of virus and E2 peptide. From an original panel of 17 mAbs, 13 mAbs were identified as E2-specific by reactivity with a BVDV 2 recombinant E2 protein expressed in insect cells. Nine E2 mAbs were observed to be virus-neutralizing. The E2 epitopes represented by the mAbs were found to be highly conserved among BVDV 2 isolates associated with hemorrhagic or severe disease in cattle. Except for one virus-neutralizing E2 mAb, the mAbs showed few or relatively weak cross-reactions with BVDV 1. Two non-neutralizing E2 mAbs were BVDV 2-specific. In contrast to BVDV 1 for which conserved neutralizing epitopes have been mapped in one immunodominant domain, the virus-neutralizing E2 mAbs produced to BVDV 2 were found to bind to highly conserved epitopes in three antigenic domains.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/genética , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/genética , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Mapeamento de Epitopos , Genótipo , Epitopos Imunodominantes/genética , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
Bovine viral diarrhea viruses (BVDV) are segregated into two genotypes, BVDV 1 and BVDV 2. Viruses within both genotypes may exist as one of two biotypes, cytopathic or noncytopathic. A highly fatal form of BVDV termed mucosal disease (MD) occurs when an animal persistently infected with noncytopathic BVDV becomes superinfected with cytopathic BVDV. In this study, we characterized a noncytopathic (BVDV2-125nc)/cytopathic (BVDV2-125c) viral pair isolated from an animal that died of MD 3 months after vaccination with modified-live BVDV1-NADL. In comparison to BVDV2-125nc, BVDV2-125c contained a 366-nucleotide insertion. The insertion was in the correct reading frame for the large open reading frame of the BVDV genome and occurred in the portion of the genome that codes for the p125 viral polypeptide. There was a 99% identity between the inserted sequences found in BVDV2-125c and sequences from the vaccine virus BVDV1-NADL. These data suggest that MD was induced after a recombination between noncytopathic BVD2-125nc and cytopathic vaccine virus BVDV1-NADL created the cytopathic virus BVDV2-125c.