Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan–bovine pestivirus primers

Abstract

The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2, respectively) and HoBi-like pestivirus (HoBiPeV) are important pathogens of cattle, and a number of reverse-transcription PCR (RT-PCR)–based assays have been developed for their detection in clinical specimens. We evaluated a newly designed set of pan–bovine pestivirus primers (BP189-389) in a gel-based RT-PCR screening test for pestiviruses in the sera of beef calves destined for export from southern Brazil. Serum samples positive for BVDV antigens by an antigen ELISA ( n = 135) were submitted to RT-PCR assays using different sets of primers, followed by nucleotide sequencing of the amplicons. RT-PCR with pestivirus primers 324-326 detected 110 positive samples: BVDV-1 ( n = 62), BVDV-2 ( n = 38), and HoBiPeV ( n = 10). A PCR using primers HCV90-368 detected 97 positive samples (64 BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2–specific primers (2F-2R) detected 45 positive samples (including 38 detected by primers 324-326 and 33 by HCV90-368); whereas a RT-PCR using HoBiPeV-specific primers (N2-R5) detected 26 positive samples (including 10 detected by primers 324-326).The assay using the primers BP189-389 detected all 135 ELISA-positive samples, including the 26 HoBiPeV detected by primers N2-R5. Our results demonstrated that primers BP189-389 compare favorably against other primer sets in the detection of bovine pestiviruses, especially HoBiPeV. This conventional PCR may be useful for efficient detection of pestiviruses in bovine sera and other specimens as well, especially in laboratories without real-time PCR equipment.