Bacterial cell surface display: a method for studying Japanese encephalitis virus pathogenicity.

Infection with Japanese encephalitis virus (JEV), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans. Recombinant Salmonella typhimurium BRD509 was constructed to display domain III of the envelope (E) protein of JEV (JEDIII) on its surface with the N-terminal domain of ice nucleation protein (INPN) as the display motif. Bacterial cell surface display was confirmed by Western blot analysis and immunohistochemical staining. Binding of recombinant INPN-JEDIII and JEDIII proteins to three mammalian cell lines was compared using a cell-binding ELISA; the human neuroblastoma cell line SK-N-SH, which had a low level of binding, was selected for further studies. The display of JEDIII on the surface of BRD509 did not significantly influence its invasiveness was confirmed by measuring released bacterial antigen using whole-cell ELISA. The relative expression of an apoptosis-related gene and total DNA damage were assessed to investigate the effects of infection on SK-N-SH cells. Compared to BRD509, infection with the recombinant bacterium reduced cell damage, suggesting that JEDIII may limit apoptosis during the early stages of JEV infection. Our studies demonstrated that it is feasible to study the pathogenesis of JEV using the approach described.