Identification and characterization of two novel linear B cell epitopes on the envelope protein of Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that can cause severe neurological disease, posing a persistent threat to both the swine industry and public health. The envelope (E) protein, a major structural protein of JEV, plays a critical role in viral entry and the induction of neutralizing antibodies, making it an important target for serological diagnosis and vaccine development. In this study, we expressed and purified recombinant JEV E protein using a prokaryotic expression system, and successfully generated two E-reactive monoclonal antibodies (mAbs) 2H9 and 5B1 from mice immunized with recombinant E protein. Using a truncated peptide approach, two novel linear B cell epitopes were identified. The minimal unit of epitope recognized by mAb 2H9 was 23LVLEGD28, and that by mAb 5B1 was 80AHNEK84. Protein sequence alignment indicated that the epitope 23LVLEGD28 was highly conserved in different JEV genotypes, while 80AHNEK84 was conserved in all genotypes except genotype V. Furthermore, structural modeling analysis showed that epitopes were located on the surface of E protein, indicating these two epitopes may be a good target for serological detection of JEV. The monoclonal antibodies generated in this study provide a key resource for developing JEV-specific serological diagnostics and studying E protein function.