Identification of three novel B cell epitopes targeting the bovine viral diarrhea virus NS3 protein for use in diagnostics and vaccine development

Bovine viral diarrhea virus (BVDV) is a major pathogen in cattle herds, widely distributed across the globe and causing significant economic losses to the cattle industry. The nonstructural protein NS3 is highly conserved across BVDV subtypes. Identifying and screening epitopes on BVDV NS3 is crucial for developing sensitive, specific diagnostic tools. In this study, we obtained three monoclonal antibodies (mAbs) against the NS3 protein: 2F7, 3E8, and 4D6. Three novel linear B-cell epitope ¹⁰⁰EYG¹⁰², ³⁸⁴FLDIA³⁸⁸, and ¹⁰⁰EYGVK¹⁰⁴ were identified through reactions of these mAbs with a series of continuous-truncated peptides and one of which a rare three-amino-acid B-cell epitope ¹⁰⁰EYG¹⁰². Critical amino acid residues were further characterized through alanine (A)-scanning mutagenesis. Sequence alignment revealed that ¹⁰⁰EYG¹⁰² and ¹⁰⁰EYGVK¹⁰⁴ were highly conserved allowing mAbs 2F7 and 4D6 to recognize all BVDV subtypes. In contrast, ³⁸⁴FLDIA³⁸⁸ was specifically conserved in BVDV-1 and BVDV-3 enabling 3E8 mAb to differential diagnosis BVDV-2 from other BVDV subtypes. Additionally, preliminary diagnostic assays for BVDV were established by western blotting and peptide-based blocking ELISA. Moreover, we observed that these mAbs could inhibit the replication of BVDV. These findings provide a theoretical foundation for developing of therapeutic strategies for nonstructural protein and accurate diagnostic procedures.