Pattama Mutthi

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.

Background and Aim: Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a global issue that affects Thai swine as well. In Thailand, PRRSV-2 predominates over PRRSV-1. The origin of PRRSV-1 transmission remains undiscovered. This study traced the source of infected pigs responsible for disease transmission among three pig-fattening farms and analyzed the spread of PRRSV-1. Materials and Methods: A total of 696 swine samples from breeding and pig-fattening farms in Thailand were screened for PRRSV using open reading frames (ORF7) reverse transcription polymerase chain reaction (RT-PCR). Positive samples were identified as PRRSV-1 using ORF5 RT-PCR. The analysis included the study of nucleotide homology, GP5 amino acid sequences, and N-linked glycosylation patterns to assess the spread of PRRSV-1 across these farms. Results: Genetic examination identified 28 PRRSV-1-positive samples, of which 13 were chosen as representatives. These strains were categorized into three groups based on breeding farm pig houses and showed distinct distribution patterns across pig-fattening farms. Group 1 included piglets transferred from pig house A to Nakhon Pathom, Chonburi, and Sa Kaeo. Groups 2 and 3 showed transfers from pig houses F and H to Chonburi and Sa Kaeo farms. All 13 PRRSV-1 strains were categorized into PRRSV-1 subtype 1/clade H. N-linked glycosylation analysis revealed that nearly all PRRSV-1 strains exhibited a conserved glycosylation pattern at amino acid positions N37, N46, and N53. This pattern is consistent with the glycosylation profile of the previous Thai PRRSV-1 subtype 1/clade H. Conclusion: The present study highlights the persistent presence of PRRSV-1 in Thai swine, which leads to sporadic outbreaks. The molecular genetic analysis identified three primary strain groups dispersed throughout the pig production system, emphasizing the importance of regular monitoring for new PRRSV strains in this herd. Understanding the PRRSV-1 distribution in swine farms is vital for veterinarians. This knowledge supports strategies for eradicating the virus and managing swine health effectively in Thailand. Keywords: porcine reproductive and respiratory syndrome virus-1, swine production system, Thailand, virus distribution.