Detection of Bovine Respiratory Syncytial Virus in Cell Cultures by nRT-PCR and Use of the Method for Virus Identification in Clinical Samples

Abstract

Valentova V., K. Kovafiaik, I. P‰ikal: Detection of Bovine Respiratory Syncytial Virus in Cell Cultures by Nested RT-PCR and Use of the Method for Virus Identification in Clinical Samples. Acta Vet. Brno 2003, 72: 115-122. One-step RT-PCR and nested PCR were used to detect bovine respiratory syncytial virus (BRSV) in infected cell cultures. Specific 984 bp and 383 bp products, selected from gene encoding the F protein using suitable primers, were amplified and, if appropriate, reamplified. The method was further used for examination of nasal swabs and blood samples collected from animals showing signs of a respiratory disease. Lactating cows and 6-8-month-old bulls from two herds were investigated. The infection by BRSV was confirmed by a) demonstration of specific fragments of BRSV genome in all animals showing signs of an acute disease, and b) indirectly by serological methods in convalescent animals. Viral RNA was detected from nasal swabs and leukocytes of affected animals. The positive PCR product obtained by nested RT-PCR from RNA isolated from leukocytes was sequenced and nucleotide and amino acid homology were 97-100% and 94.5-100% respectively, when compared with sequences from the GenBank. Considering the difficulties associated with the demonstration of BRSV in cell cultures, the amplification techniques provide an effective tool for the identification of the causative agent of bovine respiratory diseases.