Indirect ELISA Using Multi-Antigenic Dominants of 3AB and 3C Recombinant Protein to Detect Antibodies Against Senecavirus A in Pigs

Senecavirus A (SVA) causes a vesicular disease in pigs with clinical signs indistinguishable from those of other swine vesicular diseases. To enable serological differentiation infected from vaccinated animals (DIVA), we developed indirect ELISAs (iELISAs) based on recombinant non-structural proteins (NSPs). A His-tagged tandem antigen, r3AB-3C, was designed by integrating immunodominant B-cell epitopes from 3AB and 3C proteins, and was successfully expressed in Escherichia coli (E. coli) and purified alongside the individual r3AB and r3C proteins. Serological evaluation results showed that the immunoreactivity of the r3AB-3C iELISA was superior to that of r3AB, which in turn was better than r3C. The r3AB-3C and r3AB iELISAs were subsequently validated. The cut-off values were established at sample-to-positive (S/P) ratios of ≥0.2635 for the r3AB-3C iELISA and ≥0.5775 for the r3AB iELISA. The r3AB-3C iELISA demonstrated higher sensitivity for detecting infection-induced antibodies than the r3AB iELISA, despite the later seroconversion of anti-NSP antibodies compared to neutralizing antibodies. In a serosurvey, the r3AB-3C iELISA revealed seropositivity rates of 35.2% in 2023 and 22.3% in 2024. In conclusion, the r3AB-3C iELISA is a valuable serological tool for monitoring SVA infection, effectively supporting DIVA strategies.