A nested polymerase chain reaction assay to differentiate pestiviruses

Viruses that comprise the Pestivirus genus cause significant losses to the livestock industry. Based on sequence analysis, currently 4 distinct genotypes are identified of which 3 infect cattle and sheep. Distinguishing between bovine and ovine isolates by serological tests has often been difficult because of a high degree of cross reactivity. In this study, a nested polymerase chain reaction (PCR) assay was developed to identify and distinguish between bovine viral diarrhea virus (BVDV) type I, BVDV type II, as well as border disease virus (BDV) genotypes. Consensis oligonucleotide primers were designed to amplify a 826-bp product from any of the 3 pestivirus types in a reverse transcription-PCR (R-PCR). This product was subjected to a second round of nested PCR with type-specific primers which yielded DNA products of unique size characteristic for each pestivirus genotype. Using this assay, we were able to rapidly characterize several viral isolates and determine that all 3 genotypes can be found among ovine isolates.