Identification of Four Novel Group-Specific Bluetongue Virus Ns3 Protein B-Cell Epitopes

Background: The non-structural protein 3 (NS3) of bluetongue virus (BTV) is the second smaller non-structural protein produced in host cells, playing an important role in BTV trafficking and release.Results: In this study, we generated five BTV NS3-reactive monoclonal antibodies (mAbs), named 3D8, 2G9, 1B5, 4H8, and 2B12. A panel of overlapping NS3-derived peptides representing the entirety of the BTV15 NS3 protein was screened to identify linear peptide epitopes recognized by each mAb. Based on the initial screen, a series of progressively truncated peptides were produced to identify the minimal linear peptide sequence required to maintain mAb binding. We found that mAb 3D8 reacted with the motif (36)PPRYA(40), 2G9 reacted with the motif (82)AEAFRDDVRLRQIK(95), 1B5 reacted with the motif (205)YNDAVRMSF(213), 2B12 and 4H8 reacted with the motif (204)SYNDAVRMSF(213). Sequence alignments demonstrated that these linear epitopes are highly conserved among all BTV serotypes, consistent with the observation that each mAb was able to recognize cells infected with BTV1-24 serotypes tested and each identified B cell epitope was able to be recognized by BTV-infect sheep serum.Conclusion: This collection of mAbs along with defined linear epitopes may provide useful reagents for investigations of NS3 protein function and the development of BTV group-specific diagnostics.