Changes in the motifs in the D0 and SD2 domains of the S protein drive the evolution of virulence in enteric coronavirus porcine epidemic diarrhea virus.

Since 2010, highly virulent mutant GII subtype porcine epidemic diarrhea virus (PEDV) strains derived from GI subtype strains have caused significant economic losses in the pig industry. However, the molecular mechanism of PEDV virulence evolution remains unclear. It has been predicted that, compared to the S proteins of GI strains, five N-linked glycosylation sites have changed in the highly virulent GII PEDV strains. To investigate how changes in these sites affect PEDV virulence, we constructed five recombinant strains harboring the above mutation sites using the GII subtype rPEDV-S(wt) as the backbone, among which rPEDV-S(mut62), rPEDV-S(mut118), rPEDV-S(mut131), and rPEDV-S(mut722) were successfully rescued, but rPEDV-S(mut235) was not. Compared to infection with rPEDV-S(wt) (100%), infection with rPEDV-S(mut62) and rPEDV-S(mut722) resulted in lower mortality in piglets (33%), and although rPEDV-S(mut118) and rPEDV-S(mut131) resulted in high mortality (100%), death was delayed. All surviving piglets were challenged orally with rPEDV-S(wt) at 21 days post-infection. The piglets in the rPEDV-S(mut62) and rPEDV-S(mut722) groups produced high levels of IgG, IgA, and cross-protective neutralizing antibodies, which protected the piglets after rPEDV-S(wt) challenge. Furthermore, the change in the structures of the rPEDV-S(mut62) and rPEDV-S(mut722) S proteins predicted with high precision by AlphaFold 3 may be the cause of the attenuated virulence. Our data provide a unique perspective on the molecular mechanism of PEDV virulence evolution from the GI to the GII subtype and identify the targets of PED live attenuated vaccines. IMPORTANCE: The continuous emergence of novel viral variants in the current landscape poses challenges for disease prevention and control. Before 2010, PED caused by GI strains was only sporadic outbreaks and not large-scale epidemics. Since 2010, highly virulent GII strains derived from GI strains have spread worldwide and caused significant economic losses. However, the molecular mechanism underlying the differences in virulence is still unclear. In this study, the differences in the predicted glycosylation sites of the S protein between the GI and GII strains were taken as the starting point to explore the key sites responsible for the variations in PEDV virulence. The results indicate that the motifs 57ENQGVNST64 and 722NSTF725 of the S protein in the GII strains are involved in the evolution of PEDV virulence. This study provides a new perspective on the molecular mechanism of PEDV virulence evolution.