Generation and epitope mapping of two distinct neutralizing mAb subsets targeting BVDV E2: a pan-pestivirus...

Bovine viral diarrhea virus (BVDV) is an important infectious pathogen responsible for substantial economic losses in the global cattle industry. The structural protein E2 serves as a major protective antigen of BVDV and is the main target for host neutralizing immune responses. However, its antigenic structure and epitope distribution remain poorly understood. Here, five novel monoclonal antibodies (mAbs) against the E2 protein of BVDV-1 were generated using hybridoma technology, and their reactivity patterns, biological properties, and recognized epitopes were systematically characterized. As a result, one mAb, TBH033, specifically reacted with BVDV, while four mAbs, TBH002, TBH004, TBH008, and TBH009, exhibited broad-spectrum reactivity against different subgenotypes of classical swine fever virus (CSFV) and BVDV, as well as E2 proteins from eight pestivirus species (BVDV-1, BVDV-2, BVDV-3, CSFV, BDV, PPeV, GPeV, and ovIT PeV). All five mAbs exhibited high binding affinity to the E2 protein and potent cross-neutralizing activity against BVDV strains of different subgenotypes. Epitope mapping with chimeric recombinant and site-mutated E2 proteins revealed that TBH002, TBH004, TBH008, and TBH009 recognized a linear epitope in the DA domain, comprising ⁷⁰H, ⁷²R, ⁷⁴L and ⁷⁶T as critical residues; TBH033 targeted a BVDV-specific conformational epitope in the DB domain, defined by the conserved amino acid motifs ¹⁰³LCP¹⁰⁵, ¹²³GP¹²⁴. These findings enrich our understanding of the antigenic structure of the E2 protein and may facilitate the design of E2-based diagnostic tools and vaccines.