Indirect ELISA Using Multi-Antigenic Dominants of VP1, VP2, and VP3 Recombinant Protein to Detect Antibodies Against Senecavirus A in Pigs

Senecavirus A (SVA) is an emerging pathogen that poses a significant threat to the global swine industry. With the advent of SVA vaccines, there is a growing need to develop serological diagnostic methods for evaluating vaccine-induced immunity. This study successfully established an indirect enzyme-linked immunosorbent assay (iELISA) through heterologous expression of a novel VP2-VP3-VP1 tandem recombinant protein in <i>Escherichia coli</i> (<i>E. coli</i>), which was constructed by integrating B-cell epitopes from VP1, VP2, and VP3. Comparative analysis using indirect ELISA revealed that the tandem recombinant VP2-VP3-VP1 protein and VP2 exhibited superior immunoreactivity. Consequently, the iELISAs for the tandem protein and VP2 were selected for further validation. Following optimization, the cut-off for the rVP2-VP3-VP1 iELISA was set at a sample-to-positive (S/P) ratio ≥ 0.60, while that for the rVP2 iELISA was set at ≥0.53. Analysis of kinetic sera from inactivated vaccine-immunized pigs showed that the rVP2-VP3-VP1 iELISA detected seroconversion synchronously with neutralizing antibodies, earlier than anti-VP2 antibodies. Finally, a serological survey for SVA was conducted in parts of mainland China from 2023 to 2024, with the rVP2-VP3-VP1 iELISA revealing an overall seroprevalence of 20.8%. These results indicate that the established detection method can be effectively used to evaluate SVA immunity and for epidemic surveillance.