Enterovirus 71 Type And Mechanism Research Of Japanese Encephalitis Virus Into Cells

This thesis focuses on the entry mechanisms of Entero virus71(EV71) and Japanese encephalitis virus (JEV). EV71is the major pathogen that causes the recent hand foot and mouth disease (HFMD) epidemics in China. Human scavenger receptor class B2(SCARB2) is the major cellular receptor for EV71on target cells. Interactions of the EV71-SCARB2have not been fully characterized, and it has not been determined whether SCARB2serves as an uncoating receptor for EV71. We first constructed EV71single round pseudotype reporter which provided a new evaluation system for EV71neutralizing antibodies and other entry inhibitors. By comparing the efficiency of the receptor from different species including human, horseshoe bat, mouse and hamster, we demonstrated that the residues between144and151are critical for SCARB2binding to viral capsid protein VP1of EV71, and seven residues from the human receptor could convert murine SCARB2, an otherwise inefficient receptor, to an efficient receptor for EV71viral infection. We also identified that EV71binds to SCARB2via a canyon of VP1around residue Gln-172. Soluble SCARB2could convert EV71virions from160S to135S particles, in particular, under acidic conditions, indicating that SCARB2is an uncoating receptor of the virus likely in late endosome. These studies elucidated the viral capsid and receptor determinants of EV71infection and revealed a possible target for antiviral interventions. JEV is the pathogen that causes Japanese encephalitis. The entry receptor of JEV remains elusive, though more than half a century have been past since the virus was first isolated in Japan. We initially attempted to apply the split ubiquitin yeast two hybrid system to find E protein interacting partners, but didn’t get reliable results that can be validated in follow-up experiments. We then constructed a JEV chimeric virus reporter and a single round pseudotype reporter, both of them could greatly facilitate using reverse genetic approach in our study. We used the JEV chimeric virus reporter and conducted a genome-wide siRNA screen to systematically investigate the host genes involved in JEV early infection. Verification of the candidate genes is ongoing. These studies ultimately will contribute to the understanding the infection mechanism of JEV and other flaviviruses.