Mapping of three novel linear B-cell epitopes on the VP7 protein of epizootic hemorrhagic disease virus with monoclonal antibodies

Abstract Epizootic hemorrhagic disease virus (EHDV) is an important Orbivirus transmitted by culicoides midges. EHDV poses a significant threat to ruminant production worldwide. The VP7 protein is a highly conserved, group-specific antigen of EHDV, which serves as a key target for serological diagnosis. In this study, the recombinant VP7 (r-VP7) protein of EHDV-1 was expressed in an Escherichia coli expression system and used to immunize BALB/c mice. Four hybridoma cell lines secreting monoclonal antibodies (mAbs) against VP7 were successfully generated by the hybridoma technique, and named 5D1, 6A7, 7B11, and 7C4. Indirect ELISA, western blot analysis, and immunofluorescence assays demonstrated that all four mAbs specifically recognized both the r-VP7 protein and the native VP7 protein in EHDV-1-infected BHK-21 cells, with favorable reactivity and specificity. The VP7 protein was progressively truncated and expressed as a series of GST fusion proteins, and the linear B-cell epitopes recognized by these mAbs were precisely identified by western blotting. The results showed that 5D1 and 7B11 recognized the epitope 83 DYIQNLATIGVLATPEI 99 , 7C4 recognized 121 PDRQPFGYFL 130 , and 6A7 recognized 229 APVNVNNPGQ 238 . Sequence alignment and cross-reactivity assays revealed that the three epitopes were highly conserved among EHDV serotypes and showed no cross-reactivity with the VP7 proteins of African horse sickness virus (AHSV) or bluetongue virus (BTV). Three-dimensional structural analysis indicated that all three epitopes were exposed on the surface of the VP7 trimer and were located in distinct structural domains. In summary, this study successfully generated four specific mAbs against the EHDV VP7 protein and identified three novel linear B-cell epitopes, providing a foundation for the development of specific EHDV serological diagnostic methods and epitope-based vaccines.