Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

Abstract

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.