Novel B cell epitopes mapping in pD205R protein of African swine fever virus using monoclonal antibodies

BACKGROUND: African swine fever virus (ASFV) is a highly significant pathogen that causes hemorrhagic fever in domestic pigs and wild boars, resulting in substantial economic losses to the swine industry worldwide. The D205R protein (pD205R), which exhibits high similarity to the eukaryotic subunit 5 of RNA polymerase II (RPB5), has been identified as a component of the ASFV virion. However, the biological functions of pD205R remain largely unexplored. RESULTS: In this study, the recombinant pD205R (rpD205R) protein was expressed using a prokaryotic expression system, and five monoclonal antibodies (mAbs) targeting pD205R were generated. Among them, four mAbs - 5C8, 5C10, 6D5 and 9H8 - were identified as belonging to the IgG2a isotype with a kappa light chain, while mAb 3A5 was of the IgG1 isotype with a kappa light chain. The specific recognition of rpD205R by all five mAbs was demonstrated by Western blot and indirect immunofluorescence assay (IFA). Moreover, mAbs 3A5, 5C8, 5C10 and 6D5 exhibited strong reactivity with the native viral pD205R protein. Furthermore, two novel linear epitopes, spanning amino acid residues 144QEEAQEFLGR153 and 174LGGRPGDFVQ183, were identified by epitope mapping using these mAbs. Sequence alignment analysis revealed that both epitopes are conserved among the analyzed representative strains. CONCLUSIONS: In this study, five monoclonal antibodies-designated 3A5, 5C8, 5C10, 6D5, and 9H8-targeting the ASFV pD205R protein were successfully generated, resulting in the identification of two novel linear epitopes. These findings not only offer valuable monoclonal antibody tools for future investigations into the biological functions of D205R but also contribute to the development of ASFV serological diagnostic methods.