Suvarin Pavasutthipaisit

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.

Intestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 h post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens.

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9^−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9^−/− mice showed an increased loss of neuronal nuclear protein⁺ mature neurons and doublecortin⁺ neuronal precursor cells and an increase in β-amyloid precursor protein⁺ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8⁺ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air–liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.