Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1.

The bluetongue virus (BTV) NS1 protein is one of the major proteins synthesized during BTV infection and is responsible for the generation of virus-specific tubules. Although some functional and structural studies on the BTV NS1 protein have been reported, there have been no reports describing the linear B-cell epitopes recognized by humoral immune responses published to date. In this study, 25 BTV12 NS1-reactive monoclonal antibodies (MAbs) and polyclonal antisera (polyclonal antibodies, PAbs) were generated and analyzed. We identified 14 linear NS1 epitopes recognized by the PAbs and MAbs using NS1-derived peptides in an enzyme-linked immunosorbent assay. Moreover, we predicted 23 linear B-cell epitopes using the ABCpred online server which employs an artificial neural network. Analysis of the predicted and identified epitopes of NS1 demonstrated the feasibility of B-cell epitope prediction. Sequence alignments indicated that the epitopes recognized by MAbs are highly conserved among BTV serotypes, but not among the other members of the genus Orbivirus, such as the African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Chuzan disease virus (CV). Importantly, we identified specific MAbs that recognized all BTV serotypes tested as well as MAbs that recognized only BTV12, suggesting that these NS1-specific MAbs could serve as a basis for BTV diagnostic approaches. The generation and identification of NS1 protein epitopes will provide the foundation for further studies about the function and structure of NS1 and novel epitope-based vaccines.

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2.

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108-119 (domain A), a subtype I/IV-specific epitope at amino acids 211-226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231-246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131-146 and 241-256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.