Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus globally, causing serious economic losses to the global pig industry since 2010. Here, a PEDV CH/Yinchuan/2021 strain was isolated in a CV777-vaccinated sow farm which experienced a large-scale PEDV invasion in Yinchuan, China, in 2021. Our results demonstrated that the CH/Yinchuan/2021 isolate could efficiently propagate in Vero cells, and its proliferation ability was weaker than that of CV777 at 10 passages (P10). Phylogenetic analysis of the S gene revealed that CH/Yinchuan/2021 was clustered into subgroup GIIa, forming an independent branch with 2020-2021 isolates in China. Moreover, GII was obviously allocated into four clades, showing regional and temporal differences in PEDV global isolates. Notably, CH/Yinchuan/2021 was analyzed as a recombinant originated from an American isolate and a Chinese isolate, with a big recombinant region spanning ORF1a and S1. Importantly, we found that CH/Yinchuan/2021 harbored multiple mutations relative to CV777 in neutralizing epitopes (S1(0), S1(A), COE, and SS6). Homology modelling showed that these amino acid differences in S protein occur on the surface of its structure, especially the insertion and deletion of multiple consecutive residues at the S1(0) epitope. In addition, cross-neutralization analysis confirmed that the differences in the S protein of CH/Yinchuan/2021 changed its antigenicity compared with the CV777 strain, resulting in a different neutralization profile. Animal pathogenicity test showed that CH/Yinchuan/2021 caused PEDV-typified symptoms and 100% mortality in 3-day-old piglets. These data will provide valuable information to understand the epidemiology, molecular characteristics, evolution, and antigenicity of PEDV circulating in China.
