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

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.