Bovine coronavirus (BCoV): occurrence, molecular diversity and standardization of a PCR to diagnosis using stool samples of calves with and without diarrhea from municipalities of São Paulo and Minas Gerais States, Brazil / Coronavírus bovino (BCoV): ocorrência, diversidade molecular e padronização de PCR para diagnóstico a partir de amostras fecais de bezerros com e sem diarréia criados em municípios dos Estados de São Paulo e Minas Gerais, Brasil

AUTOR(ES)

Paulo Eduardo Brandão

DATA DE PUBLICAÇÃO

2004

RESUMO

Bovine coronavirus (BCoV) belongs to group 2 of the genus Coronavirus from the order Nidovirales, family Coronaviridae and causes diarrhea in newborn calves, respiratory diseases in non-newborn calves and dysentery in cows. In the present study, 203 stool samples of calves from 19 dairy farms from São Paulo and Minas Gerais States were submitted to hemagglutination/ hemagglutination inhibition test (HA/HI) to bovine coronavirus detection and a PCR assay targeted to the RNA-polymerase RNA-dependent gene of coronaviruses (PCR pol), the comparison between the two tests carried out with Kappa and Youden´s J tests. Samples positive to PCR pol were submitted to a PCR assay that amplifies a 488 base-pair fragment which corresponds to the hypervariable region of the gene coding for the S1 subunit of the S protein; the amplified fragments were submitted to DNA sequencing aiming the genealogic reconstruction of the studied samples. Rotavirus was surveyed with the PAGE test. The HA/ HI test resulted 35.47% of samples and 73.68% of farms positive to BCoV, while, according to PCR pol, 25.12% of the samples and 52.63% of the farms were positive to this virus. The comparison between the two tests produced a kappa value of -0.048 to individual results and -0.08 to the farms and Youden´s J value of -0.045 to individual results and -0.1 to the farms, showing low agreement between the two tests. Maximum parsimony genealogy with an heuristic algorithm based on sequences of the hypervariable region of the gene coding for the S1 subunit of the S protein from 15 field samples here studied, from the Kakegawa bovine coronavirus strain used as positive control and from 10 sequences retrieved from GenBank showed the existence of two genotypes in this viral species. Mean nucleotide identity between the 15 Brazilian samples was 98.34%, with mean amino acid similarity of 98%. Samples from genotype 2 showed a deletion of 18 nucleotides/ 6 amino acids inside the domain II region of the S protein. Rooted maximum parsimony tree with bredavirus as an outgroup revealed that this deletion has happened only once in bovine coronavirus genealogy. Rotavirus was found in 12.6 % of stool samples and 28.57% of the surveyed farms. These are the first results based on Brazilian strains of bovine coronavirus and contribute to molecular characterization of BCoV, to the prediction of the efficiency of immunogens and to the finding of molecular markers useful to continued epidemiologic surveys on newborn bovine diarrhea.

ASSUNTO(S)

pcr genotypes genealogia bovinos pcr diarréia coronavirus genotipos bovines genealogy corononavírus diarrhea

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