RESUMO
Bovine viral diarrhea virus (BVDV) of the genus Pestivirus is known as a common contaminant of cell culture-derived vaccines. Hog cholera virus (HCV), which is also of the genus Pestivirus and an important livestock disease in Korea, is recognized as a potential contaminant of vaccines produced in porcine cells. However, it is difficult for the National Biological Assays of korea to adequately detect contamination of these agents in biological products. For these reasons, we established rapid and sensitive methods for the detection of BVDV and HCV contamination in cell cultures and veterinary biologicals by using RT-PCR and nested PCR assays. We designed a Pestivirus primer amplifying 152 bp to detect both BVDV and HCV and a common primer amplifying 237 bp to detect only BVDV. Also, for the differentiation between BVDV type 1 and type 2, nested PCR was conducted using the amplified 237 bp PCR product, to amplify 179 bp in BVDV type 2 genome. The sensitivity of the PCR using common primer for the detection of BVDV was 400 TCID50/ml. All 6 strains of Korean BVDV isolates, 5 vaccines strains and the standard strain NADL could be detected. No reactions were observed when testing 5 types of viruses infecting pigs (HCV, TGEV, PEDV, JEV, PRRSV), 4 types infecting cattle (Akabane virus, BEFV, BCV, BRV) and 4 types infecting cats (FIP, FPL, FCV, FVR). Using this RT-PCR assay, commercial vaccines were tested and, 55 lots from 12 vaccine companies, were negative for BVDV contaminations. Same results were obtained by the immunoflourescence assay. The newly developed PCR or RT-PCR assays can be used as rapid, reliable, sensitive, and simple methods for the detection of BVDV (Pestivirus) in cell cultures, master seeds, and live viral vaccines.