Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global swine industry. Numerous modified live vaccines (MLVs) against PRRSV have been developed through the serial passage of wild-type parental strains in Marc-145 cells. However, the infectivity of these MLVs toward their primary target cell in vivo, porcine alveolar macrophage (PAM), is markedly reduced. The underlying mechanism for this decreased tropism remains unclear. In this study, we determined that the 118th residue in GP2a and the 43rd residue in GP4 play critical roles in determining viral tropism for PAM cells. Individual or combined mutations of GP2a-V118I and GP4-N43D significantly enhanced viral attachment to PAM cells. Furthermore, synthesized small peptides containing GP2a-I118 and GP4-D43 effectively blocked viral attachment to PAM cells in a dose-dependent manner, exhibiting broad-spectrum blocking effects against various PRRSV strains, including newly emerged NADC30- and NADC34-like PRRSVs. Coimmunoprecipitation (co-IP) results demonstrated that mutations or deletions at GP2a-V118 and GP4-N43 could significantly affect the binding affinity of GP2a and GP4 for the CD163 receptor, particularly the SRCR5 domain of CD163. Overall, for the first time, we identified two key residues associated with the reduced infectivity of PRRSV MLVs, which play essential roles in the attachment of PRRSV to the CD163 receptor. These findings provide novel insights into PRRSV tropism for PAM cells and reveal new targets for the development of peptide-based drugs or neutralizing antibodies against PRRSV infection.

Importance: Currently, most modified live vaccines (MLVs) against animal diseases are derived from serial passages of parental virulent viruses in heterologous animal cells. This process enhances viral adaptation to heterologous cells while significantly reducing viral infectivity to host animal cells, thereby attenuating virulence in hosts. However, the mechanisms underlying the changes in tropism of many MLVs remain largely unknown. In this study, we identified and confirmed two key residues associated with changes in tropism. Importantly, we demonstrated that small peptides can block viral binding to receptors. These findings not only provide potential targets for the development of antiviral drugs or neutralizing antibodies but also offer valuable references for studying tropism changes in other viruses.

Keywords: CD163 receptor; PRRSV; blocking peptide; cellular tropism.