Detection of bovine retrospumavirus by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detection of bovi ne retrospumavirus (bovine syncytial virus; BSV) provirus DNA. Two differen t sets of oligonucleotide primers complementary to sequences located in the gag and the pol/env gene regions were used and compared for their ability to amplify the targeted BSV sequences by PCR. The results obtained from thi s study have shown that it is possible to amplify the BSV provirus DNA sequ ences not only from total DNA of BSV-infected cell cultures, but also from total DNA of various tissues and peripheral blood mononuclear cells (PBMCs) that were collected from two rabbits experimentally infected with BSV. Sen sitivities of the PCR for amplification of BSV gag and pol/env nucleic acid sequences from cell culture total DNA were 10 ng and 10 pg of DNA, respect ively, as determined by the analysis of the amplified PCR products on ethid ium bromide-stained agarose gels. The specificity of the PCR for both prime r sets tested was confirmed when the amplified cDNA products of the expecte d size reacted positively with the corresponding virus-specific digoxigenin -labeled cDNA probes in Southern blot chemiluminescent hybridization assays . No amplification was obtained when the BSV-specific primers were used in the PCR with DNA material specific to either bovine leukemia Virus (BLV) or bovine immunodeficiency virus (BIV) provirus genomic DNA. No cross-hybridi zation was obtained when the BSV-specific cDNA probes were allowed to react with BLV or BIV provirus DNA. The PCR targeting the gag and pol/env gene r egions of the BSV provirus genome may be an alternative to conventional met hods for the confirmation of the presence of BSV in cell cultures used for virus isolation, and for the diagnosis of BSV infection from bovine periphe ral blood leukocytes. (C) 1999 Elsevier Science B.V. All rights reserved.