DETECTION AND IDENTIFICATION OF RUMINANT AND PORCINE PESTIVIRUSES BY NESTED AMPLIFICATION OF 5'-UNTRANSLATED CDNA REGIONS

Based on published gene sequences of bovine viral diarrhoea virus (BVD V) type I and classical swine fever virus (CSFV), genus- and species-s pecific primers were designed to detect and identify pestivirus cDNA s equences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, co mprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as other s of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 ca ttle isolates, including 5 typed antigenically as BVDV type II were am plified by the internal BVDV-specific primers, but not the CSFV-specif ic primers. The same result was found for other BVDV type I and II vir uses isolated from sheep and pigs. Seventy-seven CSF viruses were ampl ified by their respective internal primers. Available information stro ngly indicate that 4 CSF viruses also amplified by the BVDV-specific p rimers had been contaminated with BVDV in cell cultures. Border diseas e viruses were mostly not detected by the BVDV-specific primers, but w ere detected weakly by the CSFV-specific primer pair. Using carrier RN A for extraction of viral RNA, the sensitivity of detection of the sin gle and nested PCR was, respectively, 5 and 50 times higher than obtai ned with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified thre e earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples. Copyright (C) 1997 Elsevier S cience B.V.