The ovine respiratory syncytial virus F gene sequence and its diagnostic application.

Abstract

Ruminant respiratory syncytial viruses (RSVs) are classified into 2 subgroups, ovine RSV and bovine RSV. Although ovine RSV infects cattle, its contribution to bovine respiratory tract disease has not been established, which is an important issue for vaccine development in cattle. Diagnosis by virus isolation or serology has low or variable sensitivity and/or specificity and polymerase chain reaction (PCR) has been recommended as a rapid and sensitive technique for RSV detection. A simple procedure has been developed to detect and identify bovine and ovine RSVs. First, the nucleotide sequence of the ovine RSV fusion (F) gene was determined and compared with representative strains of bovine RSV and human RSV subgroups A and B. The ovine RSV F gene has 85 and 72-73% nucleotide identity with those of bovine RSV and human RSV, respectively. The predicted amino acid sequence of the ovine RSV F gene has 94 and 83-84% amino acid identity with those of bovine RSV and human RSV, respectively. Then PCR primers targeting a specific F gene fragment of bovine and ovine RSV were designed. The primers represented bases 85-103 and the complementary sequence to bases 510-493 of the ovine RSV F gene. A similar PCR product (426 bp) was obtained on agarose gel electrophoresis from bovine RSV and from ovine RSV. The products, however, were unique to the parent virus and could be distinguished by EcoRI or MspI restriction endonuclease cleavage. EcoRI cleaved the ovine product into 2 bands (285 and 141 bp) but failed to affect the bovine RSV PCR product. However, MspI cleaved the bovine product into 2 bands (229 and 197 bp) but had no effect on the ovine product. Also, this assay did not amplify any PCR product with human RSV. The reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction enzyme digestion is a useful and practical approach for detection and differentiation of ruminant respiratory syncytial viruses.