Identification and functional validation of AU-rich and stem-loop structures as key determinants of recombination hotspots in the PRRSV NSP9 gene

Porcine reproductive and respiratory syndrome virus (PRRSV) employs RNA recombination to generate phenotypic variants that evade host immunity, with recombination hotspots frequently observed in the NSP9 gene of field strains. This study investigates the structural determinants of recombination hotspots in the NSP9 gene by integrating bioinformatics analyses and in vitro and in vivo co-infection experiments. We identified three high-frequency recombination hotspots (7492–7624 nt, 7887–7940 nt and 9009–9464 nt) characterized by AU-rich sequences and stem-loop structures. Notably, these hotspot profiles were conserved between field-derived strains and in vitro co-infection models, supporting the use of experimental systems to highlight candidate recombination-prone contexts. To assess the functional role of RNA motifs, synonymous mutations disrupting AU-rich regions or secondary structures were engineered into the 7887–7940 nt hotspot. These mutations significantly reduced recombination rates, underscoring the contribution of local nucleotide composition and structural features to recombination dynamics. Collectively, our findings define sequence/structure contexts that shape PRRSV recombination signatures and provide a mechanistic framework that may inform efforts to improve the genetic stability of live vaccine candidates by reducing recombination propensity while acknowledging that validation in porcine target cells and/or in vivo models is required.