Amplification of strains of bovine herpesvirus 1 by use of polymerase chain reaction with primers in the thymidine kinase region

Abstract

Summary A primer pair was designed from the published nucleotide sequence of the coding region of the bovine herpesvirus 1 (bhv-1) thymidine kinase (tk) gene for use in detection of the virus by use of polymerase chain reaction (pcr) amplification of 12 bhv-1 strains (3 ATCC and 9 local isolates). A tk deletion mutant bhv-1, and 2 bhv-4 strains from ATCC were used as negative controls. One strain each of feline herpesvirus, equine herpesvirus, and bovine adenovirus, and 2 noninoculated bovine cultured cells-bovine fetal testis and Madin-Darby bovine kidney-also were examined to verify specificity of the primers. A pcr product, 183 bp long, was detected by ethidium bromide staining after agarose gel electrophoresis, when purified dna from cell cultures infected with bhv-1 strain LA was used as template. Specificity of the pcr product was confirmed by restriction digestion with Sac II enzyme and Southern blot hybridization. Amplification was detected by ethidium bromide staining of agarose gels and/or Southern blot hybridization with the radiolabeled pcr product of the LA strain in similarly prepared dna templates of 5 other bhv-1 strains, 2 obtained from ATCC and 3 of the 9 local isolates. In a modified pcr protocol, using virus suspensions treated with a nucleic add-releasing cocktail, substantial amplification was obtained for the 3 bhv-1 strains from ATCC and for all 9 local bovine herpesvirus field isolates. A tk deletion mutant bhv- 1 strain NG dltk, 2 bhv-4 strains, a feline herpesvirus, an equine herpesvirus, and a bovine adenovirus obtained from ATCC, and noninoculated bovine fetal testis and Madin-Darby bovine kidney cell cultures amplified either from purified dna or from nucleic acid-releasing cocktail-treated suspensions had negative results of ethidium bromide staining and Southern blot hybridization. These data indicate that pcr amplification, using primers in the tk gene region, is valuable for detection of bhv-1 strains grown in cell culture.