Identification of two conserved linear antigenic epitopes on the 2C protein of Senecavirus A

Abstract

Senecavirus A (SVA) is a newly emerging picornavirus threatening the global swine industry, causing vesicular disease and neonatal mortality in pigs. The non-structural protein 2C of SVA is a multifunctional virulence factor. To provide robust tools for a comprehensive study of this protein's function, we successfully generated two monoclonal antibodies (mAbs; 1F9 and 6B4) by immunizing BALB/c mice with the prokaryotically expressed 2C protein as the immunogen. Indirect immunofluorescence assays confirmed that these mAbs specifically recognized the native 2C protein. Western blot analysis further substantiated their reactivity, revealing that the recognized epitopes are linear. Both 1F9 and 6B4 were characterized as IgG1/κ isotypes. Sequence analysis of the heavy and light chain variable regions showed that the framework and complementarity-determining region (CDR) sequences were entirely distinct between the two mAbs. The antigenic epitopes recognized by 1F9 and 6B4 were precisely mapped to amino acids ¹⁶²DGYKGQF¹⁶⁸ and ³⁴LQAWINKE⁴¹, respectively, through the expression of a series of truncated forms of 2C protein. Amino acid sequence alignment of the 2C protein from global SVA strains in the GenBank database indicated that these epitopes are highly conserved. Molecular docking revealed that mAbs 1F9 and 6B4 bind to SVA 2C via hydrophobic interactions, hydrogen bonds, and salt bridges involving specific residues in their heavy and light chain CDRs. The successful development of these mAbs provides a powerful tool for the functional investigation of SVA 2C protein.