ABSTRACT
Isolates of bovine viral diarrhoea virus (BVDV) detected in serum samples of two persistently infected animals (PI) identified in a herd located in the southern state of Minas Gerais, Brazil, underwent genetic characterization trough partial nucleotide sequencing and analysis of the 5 Untranslated Region (5UTR) of the viral genome. The isolates were characterized as belonging to genotype BVDV-1, subgenotype BVDV-1b. The results of this study suggest BVDV-1b as an agent of importance in the occurrence of bovine viral diarrhoea (BVD) in the herds of the region. Moreover, the genotypic characterization of isolates of BVDV helps to better understand the epidemiology of the disease, as the genetic variability of BVDV interferes in the serological tests and has implications for the use of vaccines, whose majority is produced only with reference strains of BVDV. Therefore, the investigation on the genetic diversity of BVDV existing in Brazil is required for the improvement of the disease prevention and control measures.
Isolados do vírus da diarreia viral bovina (BVDV) detectados em amostras de soro sanguíneo de dois animais persistentemente infectados (PI), identificados num rebanho bovino localizado na região sul do Estado de Minas Gerais, Brasil, foram submetidos à caracterização genética através do sequenciamento parcial de nucleotídeos da região 5UTR do genoma viral. Os isolados foram caracterizados como pertencentes ao genótipo BVDV-1, subgenótipo BVDV-1b. Os resultados do presente estudo sugerem o BVDV-1b como um agente de importância na ocorrência da diarreia viral bovina (BVD) nos rebanhos da região. Ademais, a caracterização genotípica dos isolados do BVDV contribui para a melhor compreensão da epidemiologia da enfermidade, pois a variabilidade genética do BVDV interfere nos testes sorológicos e também possui implicações na utilização de vacinas, cuja maioria é produzida apenas com estirpes de referência do BVDV, requerendo, portanto, investigações sobre a diversidade genética do BVDV existente no Brasil para o aprimoramento das medidas de prevenção e controle da enfermidade no país.
Subject(s)
Animals , Cattle , Diarrhea/veterinary , Epidemiology , Virology , Serology , Vaccines/pharmacologyABSTRACT
A pesquisa de animais persistentemente infectados (PI) pelo vírus da diarréia viral bovina (BVDV) foi realizada em 26 rebanhos bovinos, não vacinados contra o BVDV, localizados nos Estados de Minas Gerais e São Paulo, Brasil. Utilizando uma estratégia de amostragem, de cada rebanho foram obtidas cinco amostras de sangue de bezerros, entre 6 e 12 meses de idade, e os soros sanguíneos foram submetidos ao teste de virusneutralização (VN) para o BVDV-1 e o BVDV-2. Os rebanhos que apresentaram pelo menos três das cinco amostras reagentes a um dos genótipos do BVDV, e com títulos de anticorpos superiores a 128, foram selecionados para a pesquisa de animais PI. Em três rebanhos que apresentaram tal condição, foram colhidas amostras pareadas de sangue de todos os bovinos do rebanho, com intervalo de 30 dias entre as colheitas, e o soro sanguíneo foi submetido ao teste de VN para o BVDV-1 e o BVDV-2. Nas amostras não reagentes a pelo menos um dos genótipos do BVDV e naquelas provenientes de bovinos com menos de seis meses de idade, realizou-se a pesquisa do BVDV pela reação em cadeia da polimerase precedida pela transcrição reversa (RT-PCR). Dos rebanhos analisados, foram detectados dois animais PI a partir de amostras obtidas nas colheitas pareadas provenientes de um rebanho localizado no Estado de Minas Gerais.
The research on persistently infected (PI) animals with bovine viral diarrhoea virus (BVDV) was conducted in 26 cattle herds, which were not BVDV vaccinated, located in the states of Minas Gerais and São Paulo, Brazil. Using a sampling strategy, five samples of blood were collected from 6 to 12-month-old calves of each herd, and the blood sera were tested by virusneutralization test (VN) to BVDV-1 and BVDV-2. The herds that had at least three out of five samples reacting to one of the genotypes of BVDV and antibody titers greater than 128 were selected to PI animals research. In three of the herds that matched the before-mentioned criteria, paired blood samples were collected from all its individuals considering a collection interval of 30 days. The blood sera of these samples were VN tested against BVDV-1 and BVDV-2. In samples not reacting to at least one of the BVDV genotypes and also in those collected from calves of less than six months of age, virus research was undertaken by reverse transcription polymerase chain reaction (RT-PCR). From the examined herds, two PI animals were detected in paired samples obtained from a herd located in the state of Minas Gerais.
Subject(s)
Animals , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Diarrhea Virus 1, Bovine Viral/pathogenicity , /pathogenicityABSTRACT
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both ≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
ABSTRACT
Twenty-five BVDV strains, detected in serum from persistently infected cattle from Peru (n=15) and Chile (n=10) were genetically characterized. The phylogenetic analysis based on the 5' UTR showed that all 25 strains belonged to genotype 1. Twenty-three of the strains could further be subdivided into subtype 1b, and two out of ten Chilean strains into subtype 1a. In conclusion, in total 23 out of 25 strains analyzed were of genotype 1, subtype 1b. This is the predominant BVDV subtype in many countries all over the world, including USA. The close homology with previously described strains reflects the influence of livestock trade on the diversity of BVDV circulating within and between countries and continents. Peru and Chile have imported large numbers of cattle from USA and Europe, mostly with insufficient or lacking health documentation.
Um total de 25 isolados do vírus da diarréia viral bovina (BVDV), sendo 15 originarias do Peru e 10 do Chile foram sujeitas a caracterização genética. A árvore filogenética baseada na análise da região proximal não-codificante (5'UTR) do genoma viral demonstrou que as 25 estirpes pertencem ao genótipo 1 do vírus BVD. Vinte e três destas estirpes puderam adicionalmente ser subdivididas no subtipo 1b, enquanto duas das 10 estirpes isolados provenientes do Chile foram identificadas como pertencentes ao subtipo 1a. Em conclusão, 23 de um total de 25 isolados analisados pertencem ao genótipo 1, subtipo 1b. Este é o subtipo de BVDV predominante em muitos países do mundo, incluindo os EUA. A elevada similaridade genética com isolados descritos anteriormente em outras regiões do mundo realça o papel do comércio internacional de gado no estabelecimento de diversidade genética do vírus BVD. Tanto o Peru como o Chile têm historia de importação de grandes quantidades de gado dos Estados Unidos e da Europa, no entanto sem suficiente documentação comprovativa do estado sanitário no que concerne a esta virose.
Subject(s)
Genetic Variation , Diarrhea Virus 1, Bovine Viral/isolation & purification , /isolation & purificationABSTRACT
Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.