An Inter-Subgenotypic PEDV Recombinant Harboring a Complete Structural Protein Cassette from the GIIb Lineage

Porcine epidemic diarrhea virus (PEDV) poses a persistent threat to the global swine industry through the continual emergence of variants driven by mutation and recombination. Here, we report the isolation and genomic characterization of PEDV2-5, an inter-subgenotypic recombinant isolated from clinical samples collected in Jiangxi Province, China. Phylogenetic analysis based on both the complete genome and the full-length S gene placed PEDV2-5 within the GIIb subgenotypes, clustering closely with FL201 and GD-1. Recombination detection using seven independent algorithms in RDP5, corroborated by SimPlot and phylogenetic incongruence analysis, identified PEDV2-5 as a recombinant between a GIIa major parental-like (PEDV-LY) and a GIIb minor parental-like (GD-1), generating a 7931-nucleotide fragment (nt 20060-27990) that spans the complete structural protein cassette (S, ORF3, E, M, N) and the 3'-terminal portion of ORF1b. The recombinant has replication-competent Vero-E6 cells, reaching a peak titer of 5.03 log10 TCID₅₀/mL at 36 h post-infection. Amino acid analysis revealed 98.49% identity between the PEDV2-5 S protein and the GIIb strain AJ1102, together with 13 unique substitutions and a "KKKG" insertion (residues 361-364), including two substitutions within neutralizing epitopes (A517F in COE and Y1374C in 2C10). Relative to AJ1102, the neutralizing epitopes were nearly completely conserved, whereas extensive divergence was observed against the classical GIa strain CV777. These findings highlight the importance of continued whole-genome surveillance for monitoring the emergence and spread of recombinant PEDV lineages and for informing future vaccine antigen-selection strategies.