The D389G mutation in E protein attenuates replication and virulence of Japanese encephalitis virus

Japanese encephalitis virus (JEV) is a mosquito-borne Orthoflavivirus that causes reproductive disorders in pigs and severe neurological sequelae in humans. Although genotypes I (GI) and III (GIII) have been dominant worldwide, the recent emergence of GV in South Korea underscores the urgent need to identify cross-genotype virulence sites. Through comparative genomic analysis, we identified the conserved D389 site in E protein (E-D389), presented in both GIII and GV strains but divergent in the attenuated GV-Sangju stain, as a potential virulence site. Our study showed that E-D389G mutation enhances dimerization propensity of the E protein. Compared to wild type (WT) virus, the E-D389G mutant exhibited reduced replication ability through impairing viral entry and release. Moreover, mice infected with the E-D389G mutant showed higher survival rate and reduced neuroinflammation, indicating that the mutation attenuated virulence. Notably, mosquito artificial feeding assays demonstrated that the E-D389G mutant exhibited lower infection rate and reduced viral load compared to WT in Culex mosquitoes. Taken together, our findings provide new understanding on JEV pathogenesis and contribute to the development of novel JEV vaccines.