Nsp2 replicase-mediated viral uncoating in porcine alveolar macrophages contributes to the attenuation of PRRSV-2 live attenuated vaccine

Type 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) poses a major threat to global swine production. While live attenuated vaccines (LAVs) remain the most effective countermeasure, the molecular mechanisms underlying PRRSV-2 attenuation remain enigmatic. Here, we reveal that PRRSV-2 LAVs exhibit impaired replication in their primary cellular targets-porcine alveolar macrophages (PAMs), with viral uncoating defects being the critical replication barrier. Mechanistically, we identified nonstructural protein 2 (nsp2) as the key viral determinant orchestrating this attenuation phenotype. Strikingly, the substitution of the nsp2 from the vaccine strain into a highly pathogenic PRRSV (HP-PRRSV) significantly attenuated virulence in piglets while maintaining immunogenicity. The chimeric virus elicited robust protective immunity against HP-PRRSV challenge. Our findings elucidated that nsp2-mediated viral uncoating contributes to PRRSV-2 LAV attenuation and established a proof-of-concept strategy for rational PRRSV-2 vaccine design.IMPORTANCELive attenuated vaccines (LAVs) are predominantly used for the management of PRRSV infection; however, limited knowledge exists regarding the mechanisms underlying PRRSV attenuation. Enhancing our understanding of the mechanism by which viruses are attenuated would accelerate the development of optimal live attenuated vaccines against PRRSV. In the present study, we discovered that commercial PRRSV LAVs failed to uncoat inside porcine alveolar macrophages, thereby identifying a novel mechanism by which these LAVs achieve attenuation. Notably, we identified nsp2, a virion protein, as a key factor contributing to the attenuation of PRRSV. Furthermore, we demonstrated that the substitution of the nsp2-coding region with its counterpart derived from a commercial LAV enabled the rapid attenuation of highly virulent strains while providing effective protection against subsequent challenges. Our findings elucidated the feasibility of converting virulent PRRSV into an attenuated vaccine candidate in a timely manner.