Validation of a Broad-Range Conventional RT-PCR Assay for Pestivirus Surveillance and Molecular Detection of BVDV-2a...

The genus Orthopestivirus, within the family Flaviviridae, includes important pathogens such as classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV-1 and BVDV-2), and border disease virus (BDV). Although Costa Rica is endemic for BVDV, it remains officially free of CSFV and BDV, highlighting the need for reliable molecular tools for pestivirus surveillance and differential diagnosis in swine populations. This study validated a conventional broad-range in-house RT-PCR assay targeting the 5' untranslated region (5'UTR) of pestiviruses, a genomic region that is highly conserved among members of the genus Orthopestivirus. A wide variety of PCR-based assays have been developed for pestivirus detection; however, many are designed specifically for the diagnosis of bovine pestiviruses, such as bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV) in cattle. In contrast, the assay described in this study was standardized and validated to detect multiple pestivirus species, thereby providing a useful tool for surveillance and diagnostic applications across different host species. A total of 66 reference samples were analyzed to determine diagnostic sensitivity and specificity, while analytical sensitivity, repeatability, reproducibility, and selectivity were also evaluated. Additionally, pestivirus surveillance was conducted from 2014 to 2025. The assay showed a limit of detection of 1.2 copies/µL for CSFV and 11.7 copies/µL for BVDV, with diagnostic sensitivity and specificity of 98% and 100%, respectively. A total of 1072 surveillance samples were tested, including 11 bovine and 1061 porcine samples. Two porcine samples collected in 2015 were positive for pestivirus and clustered within the BVDV-2a group, representing the first molecular detection of BVDV-2a in pigs in Costa Rica. Circulation of BVDV-1b in cattle was also confirmed. The assay demonstrated satisfactory performance as a broad-range pestivirus surveillance tool and may support differential diagnosis in CSFV-free countries when combined with sequencing confirmation for pestivirus species identification.