# 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

## TL;DR

This study develops a new diagnostic method to detect and classify PRRSV-2 in Thailand, revealing new virus sublineages and improving disease monitoring.

## Contribution

A novel nested RT-PCR assay combined with ORF5 phylogenetic analysis for enhanced detection and classification of PRRSV-2 in Thailand.

## Key findings

- Nested ORF7 RT-PCR showed high specificity and 100% agreement with real-time RT-PCR.
- Thai PRRSV-2 strains were classified into five lineages and five sublineages, including newly reported sublineages L8C and L9D.
- A lineage shift from L8E to L10 was observed as the predominant circulating strain in Thailand.

## Abstract

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.

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.

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.

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.