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

## TL;DR

A new ELISA test using a recombinant protein detects antibodies against Senecavirus A in pigs, aiding in vaccine monitoring and outbreak tracking.

## Contribution

Development of a novel indirect ELISA using a VP2-VP3-VP1 tandem recombinant protein for reliable detection of SVA antibodies.

## Key findings

- The VP2-VP3-VP1 tandem recombinant protein showed high specificity and sensitivity in detecting SVA antibodies.
- The assay detected seroconversion earlier than anti-VP2 antibodies in vaccinated pigs.
- A serological survey in China revealed a 20.8% seroprevalence of SVA.

## Abstract

Senecavirus A (SVA) is defined as an emerging viral agent responsible for inducing vesicular lesions in swine. The recent development of SVA vaccines has created a need for reliable methods to evaluate post-vaccination immune responses for diagnostic and monitoring purposes. Here, we developed a serological assay based on a designed tandem antigen, VP2-VP3-VP1, as its core component. The assay demonstrated high specificity and sensitivity, confirming its suitability for assessing immune responses following vaccination. When applied to a panel of more than 3800 porcine serum samples, the assay demonstrated high reliability and confirmed its utility for large-scale serological surveillance. In summary, this newly developed serological tool is valuable for the reliable monitoring of vaccination efficacy and outbreak surveillance.

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 Escherichia coli (E. coli), 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.