A Spanish Porcine Reproductive and Respiratory Syndrome Virus 1 Strain Is Highly Virulent in Pigs Under Experimental Conditions

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most important infectious diseases affecting the global pig industry. The emergence of highly pathogenic PRRS virus 1 (PRRSV-1) strains, first detected in northeastern Spain in 2020, poses new challenges for effective disease control. Despite their severe impact under field conditions, a comprehensive experimental characterization of these virulent strains has been lacking. In this study, we analyzed the genetic characteristics of the Lleida 029_22 strain, collected during a severe PRRSV-1 outbreak, and evaluated its pathogenicity in pigs. Phylogenetic analysis revealed that the Lleida 029_22 strain belongs to a new clade describing multiple strains isolated during a PRRSV-1 Rosalia-related outbreak, with the highly pathogenic Italian strain PR40 identified as a common ancestor. <i>In vitro</i>, this strain replicated efficiently in porcine alveolar macrophages and PAM-KNU cells, but not in MARC-145 cells. To characterize the pathogenicity of this strain, 8-week-old piglets were inoculated with a 2 × 10⁵ TCID₅₀ dose of the Lleida 029_22 strain via two inoculation routes-intramuscular (IM) and intranasal (IN). The IM-infected pigs exhibited high levels of viremia and virus shedding, with 100% mortality recorded by 14 days post-infection (dpi). These animals also showed significantly elevated levels of pro-inflammatory cytokines, particularly IL-6, and severe histopathologic lung lesions, correlating with the observed severe disease outcome. In contrast, IN-infected pigs exhibited lower mortality (30%) and moderate clinical signs, with the surviving pigs fully recovering by the end of the 63-day observation period. These pigs also exhibited prolonged viremia and viral shedding, with lower levels of pro-inflammatory cytokines and neutralizing antibodies from 28 dpi. The IN infection closely mimicked the disease outcomes observed in Spanish farms experiencing outbreaks due to the high-virulence Rosalia strain, while the IM infection highlighted the risks of iatrogenic transmission.