Epitope Mapping of Senecavirus A 3A Protein Using Monoclonal Antibodies

Senecavirus A (SVA), an emerging pathogen causing vesicular disease in pigs, poses a significant threat to the swine industry. The nonstructural protein 3A of SVA plays an essential role in the viral replication cycle. In this study, we immunized mice with the prepared SVA 3A protein and produced two monoclonal antibodies (mAbs), AG4 and 2F3. MAb AG4 showed specific reactivity to the linear and conformational 3A protein, whereas mAb 2F3 did not recognize linear epitope of 3A protein. Through truncated 3A protein expression and alanine mutation analysis, we identified ¹SPNEND⁶ as the minimal motif recognized by mAb AG4, with Asn³ being the critical residue. Additionally, we demonstrated that mAb 2F3 failed to recognize the SVA mutant with the ⁷⁵QEETEG⁸⁰ deletion in 3A protein, indicating that ⁷⁵QEETEG⁸⁰ constitutes an essential epitope for mAb 2F3. Further deletion analysis confirmed that ⁷⁵QE⁷⁶ is the crucial motif for mAb 2F3 recognition. Moreover, we found that ¹SPNEND⁶ and ⁷⁵QEETEG⁸⁰ are highly conserved among different SVA strains and are exposed on the surface of the 3A protein. This study contributes to further explore the function of SVA 3A protein and develop diagnostic tools for SVA detection.