Novel linear B-cell epitopes mapping with monoclonal antibodies to VP3 protein from bluetongue virus serotype 1

Bluetongue is an infectious disease affecting domestic and wild ruminants that is caused by bluetongue virus (BTV). VP3 is the major component of the BTV inner capsid and a structural protein that is essential for viral maturation. The B-cell epitopes on BTV-1 VP3 that are recognized by the humoral immune response remain largely uncharacterized. In this study, we expressed and purified recombinant BTV-1 VP3 in Escherichia coli and used it to immunize BALB/c mice. Hybridoma technology was used to generate eight monoclonal antibodies (mAbs): 3A5, 3B2, 3E6, 3H5, 3B4, 3C5, 3D3, and 3E1. The B-cell epitopes recognized by these mAbs were mapped using a series of overlapping truncated peptides expressed as glutathione S-transferase fusion proteins. Two linear epitopes were identified: ⁷QRPERIKTT¹⁵, recognized by mAbs 3B4, 3C5, 3D3, and 3E1, and ¹¹⁹TEDEPEKFYS¹²⁸, recognized by mAbs 3A5, 3B2, 3E6, and 3H5. Cross-reactivity analysis confirmed that these mAbs were specific to BTV1 VP3 under the test conditions, with the limitation that cross-reactivity validation was performed only against EHDV-2 VP3. This study identified key antigenic epitopes on BTV-1 VP3 under test conditions, providing structural studies, diagnostic tool development, or viral assembly research. These findings expand the available repertoire of BTV-1 VP3-specific monoclonal antibodies and define two linear B-cell epitope regions within VP3, providing a foundation for future structural studies, diagnostic tool development, and investigations into BTV core assembly.