Vet World Vol.18 September-2025 Article - 24
Research Article
Veterinary World, 18(9): 2850-2866
https://doi.org/10.14202/vetworld.2025.2850-2866
Nested open reading frame (ORF) 7 reverse transcription polymerase chain reaction and ORF5 phylogenetic refinement for enhanced detection and genetic classification of porcine reproductive and respiratory syndrome virus-2 in Thailand
1. Department of Preclinical Sciences, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, 10530, Thailand.
2. Department of Clinical Sciences, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, 10530, Thailand.
3. Department of Science, Technology and Innovation, Faculty of Science, Chulabhorn Royal Academy, Bangkok, 10210, Thailand.
4. Faculty of Veterinary Medicine, Rajamangala University of Technology Tawan-ok, Chonburi, 20110, Thailand.
5. Department of Pathology, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, 10530, Thailand.
Background and Aim: Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major economic threat to the global swine industry, causing reproductive losses and severe respiratory illness. Accurate and cost-effective diagnostic tools are essential for timely detection and genetic monitoring, particularly in resource-limited settings. This study aimed to (i) establish a nested reverse transcription polymerase chain reaction (RT-PCR) assay targeting the open reading frame 7 (ORF7) gene to detect and differentiate PRRSV-1 and PRRSV-2, and (ii) refine the genetic classification of PRRSV-2 strains circulating in Thailand through ORF5-based phylogenetic analysis.
Materials and Methods: A nested RT-PCR assay was developed using external primers for general PRRSV detection and internal primers specific to PRRSV-1 and PRRSV-2. Analytical specificity was assessed against modified-live vaccines, clinical isolates, and heterologous swine viruses (swine influenza virus and foot-and-mouth disease virus). Diagnostic accuracy was evaluated using 96 clinical serum samples and compared with a commercial real-time RT-PCR kit. To confirm genotyping capability, ORF7-positive samples underwent ORF5 sequencing and phylogenetic analysis. In addition, 386 complete ORF5 sequences (2000–2023) from Thai isolates and global references were analyzed using maximum likelihood methods to refine lineage and sublineage classification.
Results: The nested ORF7 RT-PCR assay demonstrated high specificity without cross-amplification and achieved 100% concordance with real-time RT-PCR, confirming its diagnostic reliability. Among the clinical samples, PRRSV-1, PRRSV-2, and mixed infections were successfully detected. Sequencing confirmed strain identities and revealed close similarity with both endemic and vaccine-related strains. Phylogenetic analysis classified Thai PRRSV-2 strains into five lineages (L1, L5, L8, L9, L10) and five sublineages (L1I, L5A, L8C, L8E, L9D). Notably, this study is the first to report sublineages L8C and L9D in Thailand, while also documenting a lineage shift from L8E to L10 as the predominant circulating strain.
Conclusion: The integration of nested ORF7 RT-PCR with ORF5-based phylogenetic analysis provides a sensitive, affordable, and reliable diagnostic platform for PRRSV detection and genetic classification. These findings enhance understanding of PRRSV-2 diversity in Thailand, highlight emerging sublineages, and underscore the importance of continuous molecular surveillance to inform vaccine strategies and disease control policies.
Keywords: molecular diagnostics, nested reverse transcription polymerase chain reaction, open reading frame 5 phylogeny, open reading frame 7, porcine reproductive and respiratory syndrome virus, Thailand.
How to cite this article: Jantafong T, Karnbunchob N, Tanomsridachchai W, Mutthi P, and Pavasutthipaisit S (2025) Nested open reading frame (ORF) 7 reverse transcription polymerase chain reaction and ORF5 phylogenetic refinement for enhanced detection and genetic classification of porcine reproductive and respiratory syndrome virus-2 in Thailand, Veterinary World, 18(9): 2850-2866.
Received: 10-05-2025 Accepted: 19-08-2025 Published online: 23-09-2025
Corresponding author: E-mail:
DOI: 10.14202/vetworld.2025.2850-2866
Copyright: Jantafong, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
CITATIONS
- Nested open reading frame (ORF) 7 reverse transcription polymerase chain reaction and ORF5 phylogenetic refinement for enhanced detection and genetic classification of porcine reproductive and respiratory syndrome virus-2 in Thailand
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