NSP9 is a key virulence determinant in highly pathogenic PRRSV-mediated thymic injury via synergistic activation of apoptotic and metabolic pathways.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) induces severe thymic atrophy, contributing to immunosuppression in infected piglets. This study investigated the roles of viral nonstructural proteins NSP9 and NSP10 in thymic pathogenesis using chimeric viruses (HC9 and HC10) generated by replacing NSP9/NSP10 of the HP-PRRSV HuN4 strain with those from the classical CH-1a strain. In vitro replication was significantly affected by these swaps, with NSP9 showing a more pronounced effect. In vivo replication kinetics, pathogenicity, and thymus damage were analyzed in piglets inoculated with the HuN4 strain or the chimeric strains. The study found that NSP9 and NSP10 are closely associated with PRRSV replication efficiency and pathogenicity, with NSP9 having a greater impact on thymus atrophy and both NSP9 and NSP10 playing a key role in inducing thymocytes apoptosis. Transcriptomic analysis revealed that HuN4 infection significantly upregulated genes associated with apoptosis, inflammatory responses, and metabolic pathways (e.g., NF-κB, PI3K-Akt, and p53 signaling), while HC9 showed attenuated effects. Flow cytometry confirmed HuN4-induced depletion of CD4+CD8+ thymocytes and dysregulated surface marker expression (CD4). TUNEL assays and apoptosis-related gene profiling further implicated NSP9 in activating both intrinsic and extrinsic apoptotic pathways. Notably, metabolic pathway enrichment suggested crosstalk between apoptosis and energy sensing (e.g., AMPK-mTOR). These findings highlight NSP9 as a critical virulence factor driving thymic atrophy through synergistic immune hyperactivation, apoptotic cascades, and metabolic reprogramming, providing novel insights for PRRSV vaccine design and immunomodulatory strategies.