Emergence of a Highly Virulent Porcine Epidemic Diarrhea Virus (PEDV) G2c Subtype in China: Isolation, Genetic and Pathogenic Characterization, and Cross-Neutralizing Antibody Response.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus causing high mortality in neonatal piglets, continues to threaten global swine industries. Frequent mutations in the spike (S) protein of PEDV, particularly in emerging variants, have substantially compromised commercial vaccine efficacy. Despite the emergence of G2c variants dominating recent epidemics, comprehensive studies integrating viral isolation, phylogenetics, structural modeling, cross-neutralizing antibody response, and pathogenicity assessment remain insufficient. In this study, we successfully isolated a G2c strain (AHCZ02) and obtained 69 S gene sequences from nine provinces during 2021-2024. Phylogenetic analysis identified G2c variants as predominant (69.57%, 48/69) in current outbreaks. Structural comparisons revealed four G2c-specific substitutions (N139D, I287M, F345L, and L998M) inducing conformational changes in critical S domains compared to G2a/G2b strains, potentially disrupting immune recognition. The results of serum neutralizing antibody (nAb) test using the AHCZ02 strain showed that G2c-based feedback exposure strategies elicited 3.9-fold higher geometric mean titers (GMTs) than S-INDEL-based approaches. Furthermore, feedback exposure strategies of G2c (GMT = 480-1893) showed 12- to 189.3-fold higher neutralizing activity versus conventional vaccines (GMT = 10-40). Pathogenicity assessment in neonatal piglets revealed 100% mortality within 66 h post-AHCZ02 inoculation, accompanied by hallmark clinical manifestations including profuse watery diarrhea, rapid weight loss, and severe jejunal villus atrophy. Collectively, these findings provide evidence that G2c variants have developed S protein modifications associated with diminished vaccine efficacy, underscoring the need for next-generation vaccines incorporating G2c-specific antigenic determinants, and strengthened virological surveillance systems to effectively monitor and respond to PEDV evolutionary dynamics.