Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typingruminant pestiviruses: Bovine viral diarrhea viruses and border disease virus

A nested reverse transcription (RT) polymerase chain reaction (PCR) assay w as evaluated for differentiating reference bovine viral diarrhea virus (BVD V) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BD V), The detection level of this assay was compared to viral infection in ce ll culture, The PCR assay was used to distinguish 3 ruminant pestiviruses, types 1 and 2 BVDV, and type 3 BDV, The consensus (first) PCR assay detecte d all 3 ruminant pestiviruses, a result of the shared sequence homology, Th e consensus PCR product was subjected to a second (nested) PCR which used t ype-specific primers. The nested PCR was able to differentiate the 3 rumina nt pestiviruses, Viral stocks of BVDV were diluted 10-fold and processed fo r the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compar ed to viral infectivity in cell culture based on identical volumes of the s ystem tested (cell culture assay and processing for RNA). The RT-PCR type-s pecific assay differentiated BVDV laboratory reference strains (12), diagno stic laboratory isolates (15), 2 MLV BVDV vaccine strains, and a BDV strain . The 30 ruminant pestiviruses typed included: (1) 27 reference strains and diagnostic laboratory isolates; 18 cytopathic (CP) type 1 strains, 3 CP ty pe 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strain s; (2) 2 MLV strains, type 1; and (3) 1 CP BDV type 3, The PCR assay had a detection limit of 10 TCID50/0.025 mk of virus when 3 separate BVDV were te sted. This 2 step RT-PCR assay would be useful for the typing of ruminant p estiviruses, particularly BVDV isolates from the diagnostic laboratory.