Abstract
Porcine epidemic diarrhea virus (PEDV), a highly virulent enteric coronavirus, induces severe watery diarrhea and mortality in suckling piglets. The spike (S) protein, a critical mediator of viral entry, undergoes extensive N-linked glycosylation. To elucidate the functional significance of these post-translational modifications, we employed a reverse genetics system to generate 19 recombinant PEDV strains with single-site mutations at predicted N-glycosylation sites. In vitro experiments revealed that mutations at residues N118, N216, N726, N1232, and N1249 significantly attenuated viral replication and reduced plaque size. Our data demonstrated that these mutations impaired viral attachment and internalization. Importantly, in vivo pathogenicity assays in piglets indicated that the N1232Q and N1249Q mutants presented minimal faecal viral shedding and no clinical symptoms, suggesting their potential as live attenuated vaccine candidates. These findings underscore the critical role of S protein glycosylation in PEDV infectivity and virulence, providing a molecular basis for rational vaccine design.