The glycosylation variant at residue 381 of the spike protein contributes to virulence shifts in porcine epidemic diarrhea virus during both natural field transmission and laboratory cell passaging with poor cross-protection.

UNLABELLED: The virulence and immunogenicity of porcine epidemic diarrhea virus (PEDV) vary during field circulation and cell culture passage (such as when the GI CV777 strain is attenuated through serial passaging). This study revealed that the glycosylation site mutation at position 381 (N381K) of the S protein is associated with these phenomena. Compared with piglets inoculated with P13 virus, piglets inoculated with P100 (N381K) of virulent GX223 exhibited delayed diarrhea, viral shedding, and mortality. Using the virulent rCH/SX/2016-SHNXP strain (rPEDV-Swt) as the backbone, we generated rPEDV-SN381K. While both the wild-type and mutant strains showed similar growth in vitro and in 2-day-old piglets, rPEDV-SN381K caused milder diarrhea and lower mortality. In 5-day-old piglets, the mutant strain also induced delayed viral shedding and milder diarrhea. At 21 days after post-infection, all the piglets were challenged with the parental strain. The pigs in both the rPEDV-SN381K and rPEDV-Swt groups produced high IgA/IgG levels, but the rPEDV-SN381K-inoculated piglets presented higher fecal viral loads and lower neutralizing antibody titers against the parental strain. Molecular modeling suggests that N381K alters antigenic epitope interactions, which may affect virulence and immunogenicity. While this study has the limitation of a relatively small sample size in the animal studies, the results collectively demonstrate that S protein glycosylation mutations influence PEDV virulence and contribute to reduced cross-protective vaccine efficacy, offering important insights for PEDV pathogenesis research and vaccine development.

IMPORTANCE: Porcine epidemic diarrhea virus (PEDV) continues to cause substantial economic losses in the global swine industry, with emerging strains challenging existing vaccine strategies. This study identifies the N381K glycosylation site mutation in the S protein of PEDV as a factor involved in variations in virulence during natural transmission and laboratory adaptation. Crucially, the mutant induces suboptimal neutralizing immunity against the prevalent strain, revealing a mechanism by which classical-strain vaccines may provide limited protection against currently circulating strains. Our findings reveal how a single glycan modification modulates both pathogenicity and immunogenicity, providing critical insights for the development of effective vaccines against circulating PEDV variants.