Abstract
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. In vitro, 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 × 10(5) TCID(50) 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.