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

• Published in: Canadian journal of veterinary research Vol. 63; no. 4; pp. 276 - 281
• Main Authors: Fulton, R.W, d'Offay, J.M, Saliki, J.T, Burge, L.J, Helman, R.G, Confer, A.W, Bolin, S.R, Ridpath, J.F
• Format: Journal Article
• Language: English
• Published: Canada 01-10-1999
• Subjects: Animals; assays; Biological Assay; Border disease virus; Border disease virus - classification; Border disease virus - genetics; Bovine viral diarrhea virus; Cattle; Cattle Diseases - classification; Cattle Diseases - diagnosis; Cattle Diseases - virology; cultured cells; Diagnosis, Differential; Diarrhea Viruses, Bovine Viral - classification; Diarrhea Viruses, Bovine Viral - genetics; DNA probes; live vaccines; polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA-directed DNA polymerase; strain differences
• ISSN: 0830-9000

A nested reverse transcription (RT) polymerase chain reaction (PCR) assay was evaluated for differentiating reference bovine viral diarrhea virus (BVDV) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BDV). The detection level of this assay was compared to viral infection in cell 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 detected all 3 ruminant pestiviruses, a result of the shared sequence homology. The consensus PCR product was subjected to a second (nested) PCR which used type-specific primers. The nested PCR was able to differentiate the 3 ruminant pestiviruses. Viral stocks of BVDV were diluted 10-fold and processed for the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compared to viral infectivity in cell culture based on identical volumes of the system tested (cell culture assay and processing for RNA). The RT-PCR type-specific assay differentiated BVDV laboratory reference strains (12), diagnostic 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 type 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strains; (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 mL of virus when 3 separate BVDV were tested. This 2 step RT-PCR assay would be useful for the typing of ruminant pestiviruses, particularly BVDV isolates from the diagnostic laboratory.