Abstract
Since 2010, millions of piglets have died from porcine epidemic diarrhea virus (PEDV) variant strains. Compared with classical strains, variants exhibit enhanced virulence and immune evasion capacity, rendering classical strain-based vaccines poorly effective. However, the critical mutants responsible for increased pathogenicity and immune evasion remain unclear. This study aims to identify the key mutations that drive humoral immune evasion in PEDV variants and to elucidate further the underlying molecular mechanisms. Cross-neutralization assays and recombinant virus screening identified the 894-993 amino acid (aa) mutation region of the S2 subunit as a key determinant of humoral immune evasion in variants. The challenge experiments in piglets demonstrated that the 894-993 aa mutation region plays a critical role in determining variants' virulence. Subsequent vaccination-challenge experiments further clarified the pivotal contribution of the 894-993 aa mutation region in the humoral immune evasion in vivo. In addition, the 894-993 aa region determines the membrane fusion characteristics, enabling variants to resist neutralizing sera through cell-to-cell transmission. Moreover, structural analyses revealed that mutations in this region altered the surface electrostatic potential and increased hydrophobicity and rigidity of the S protein. The findings of this study are the first to demonstrate that the 894-993 aa mutation region of the S2 subunit drives humoral immune evasion, modulates the virulence and membrane fusion of PEDV variant strains, and provides a theoretical foundation for the formulation of new prevention and control strategies.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) variant strains pose a serious threat to piglet health worldwide. Despite the availability of commercial vaccines developed based on classical PEDV strains, they have shown limited efficacy against newly emerging variants. Moreover, variant strains exhibit varying degrees of genomic mutations compared to classical strains, and the regulatory effects of these mutations on viral biology have not been systematically studied. In this study, we identify the 894-993 amino acid (aa) region within the S2 subunit as a key determinant of humoral immune evasion in variants. Mutations in this region were shown to reduce neutralization sensitivity, alter membrane fusion activity, and increase the structural rigidity of the S protein. These findings greatly enhance our understanding of the biological characteristics of PEDV variants and provide a new potential strategy and important theoretical support for viral control and vaccine development.