A novel PEDV-specific linear B-cell epitope evades cross-reactivity with TGEV and PDCoV.

The high incidence of swine enteric coronavirus co-infections necessitates highly specific diagnostic tools targeting key viral antigens, such as the porcine epidemic diarrhea virus (PEDV) nucleocapsid (N) protein. In this study, we generated monoclonal antibodies (mAbs) against the recombinant PEDV N protein and mapped a novel, highly conserved linear B-cell epitope, 272-ERDLKDIPEWR-282. To define the molecular basis of this interaction, alanine scanning mutagenesis identified residues Leu275, Lys276, Asp277, Ile278, Glu280, Trp281, and Arg282 as the core motif essential for mAb recognition. Furthermore, structural modeling predicted that this epitope adopts an exposed α-helix and flexible loop conformation. Crucially, sequence alignments revealed poor conservation of this epitope across other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV). Subsequent in vitro validation via indirect immunofluorescence assays (IFA) and ELISA confirmed that the targeting mAb, 1D1, exclusively recognizes PEDV antigens, exhibiting no detectable cross-reactivity with TGEV or PDCoV. These findings elucidate the fine antigenic profile of the PEDV N protein and highlight mAb 1D1 and its corresponding epitope as promising candidates for the differential diagnosis of major swine enteric coronaviruses.