Establishment and Application of a Novel Protein Microarray for Serological Detection and Differentiation of Senecavirus A

Senecavirus A (SVA) is an emerging swine pathogen that causes vesicular disease, which presents clinically indistinguishable signs from other vesicular diseases. To enable differentiation of infected from vaccinated animals (DIVA), we developed a novel protein microarray for dual serological detection of antibodies against SVA structural (VP2‐VP3‐VP1) and non‐structural (3AB‐3C) proteins. The assay was based on two novel His‐tagged tandem antigens, designed from immunodominant B‐cell epitopes, which were expressed in Escherichia coli ( E. coli ), purified, and spotted onto a poly(dimethylsiloxane) (PDMS) substrate. Results were quantified by spot gray values to calculate sample‐to‐positive (S/P) ratios, with cut‐off values set at S/P ≥0.651 for VP2‐VP3‐VP1 and S/P ≥0.607 for 3AB‐3C. The microarray successfully differentiated inactivated‐vaccine‐immunized animals (positive only for VP2‐VP3‐VP1) from live SVA‐challenged animals (positive for both antigens). In live SVA‐challenged pigs, seroconversion to the structural protein antigen VP2‐VP3‐VP1 occurred 4 days earlier than the non‐structural protein antigen 3AB‐3C, identifying it as a sensitive early diagnostic marker. Clinical validation demonstrated 97.5% concordance with the virus neutralization test (VNT), confirming the microarray as a reliable, high‐throughput tool for DIVA serological testing.