{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:39:07Z","timestamp":1785425947487,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:00Z","timestamp":1783728000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the 2024 PROCINORTE call for training and research proposals on African swine fever (ASF) and Classical swine fever virus in the Americas"},{"name":"Inter-American Institute for Cooperation on Agriculture through the 2012 Research and Internship Assistance Program for Agriculture in the Americas"},{"id":[{"id":"https:\/\/ror.org\/01vkp3w06","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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\u2032 untranslated region (5\u2032UTR) 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\/\u00b5L for CSFV and 11.7 copies\/\u00b5L 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.<\/jats:p>","DOI":"10.3390\/v18070762","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T09:51:09Z","timestamp":1783936269000},"page":"762","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Validation of a Broad-Range Conventional RT-PCR Assay for Pestivirus Surveillance and Molecular Detection of BVDV-2a in Pigs in Costa Rica"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0070-6746","authenticated-orcid":false,"given":"Bernal","family":"Le\u00f3n","sequence":"first","affiliation":[{"name":"LSE, Laboratorio Nacional de Servicios Veterinarios, Servicio Nacional de Salud Animal, Servicio Nacional de Salud Animal, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Idania","family":"Chac\u00f3n","sequence":"additional","affiliation":[{"name":"LSE, Laboratorio Nacional de Servicios Veterinarios, Servicio Nacional de Salud Animal, Servicio Nacional de Salud Animal, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eunice","family":"V\u00edquez","sequence":"additional","affiliation":[{"name":"LSE, Laboratorio Nacional de Servicios Veterinarios, Servicio Nacional de Salud Animal, Servicio Nacional de Salud Animal, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga","family":"Aguilar","sequence":"additional","affiliation":[{"name":"LSE, Laboratorio Nacional de Servicios Veterinarios, Servicio Nacional de Salud Animal, Servicio Nacional de Salud Animal, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guisella","family":"Chaves","sequence":"additional","affiliation":[{"name":"LSE, Laboratorio Nacional de Servicios Veterinarios, Servicio Nacional de Salud Animal, Servicio Nacional de Salud Animal, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9621-3972","authenticated-orcid":false,"given":"Tamara","family":"Solorzano","sequence":"additional","affiliation":[{"name":"Laboratory of Virology, Tropical Disease Research Program, School of Veterinary Medicine, Universidad Nacional, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9544-5285","authenticated-orcid":false,"given":"Carlos","family":"Jim\u00e9nez","sequence":"additional","affiliation":[{"name":"Laboratory of Virology, Tropical Disease Research Program, School of Veterinary Medicine, Universidad Nacional, Heredia 40104, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Pasick","sequence":"additional","affiliation":[{"name":"Formerly of the Canadian Food Inspection Agency, National Centre Foreign for Animal Disease, 1015 Arlington Street, Winnipeg, MB R3E 3M4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8644-3560","authenticated-orcid":false,"given":"Llilianne","family":"Ganges","sequence":"additional","affiliation":[{"name":"WOAH Reference Laboratory for Classical Swine Fever, Institute of Agrifood Research and Technology, Centre de Recer\u00e7a en Sanitat Animal (CReSA), 08193 Bellaterra, Spain"},{"name":"IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), 08193 Bellaterra, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3026","DOI":"10.1038\/s41564-025-02134-0","article-title":"Taxonomic Expansion and Reorganization of Flaviviridae","volume":"10","author":"Simmonds","year":"2025","journal-title":"Nat. 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