Non-Dominant Genotypes (GII, GIV and GV) of Japanese Encephalitis Virus Exhibit an Elevated Evolutionary Rate in Nature

Numerous studies have demonstrated that the Japanese encephalitis virus (JEV) is classified into five genotypes. Historically, JEV GIII and GI were the dominant strains before and after the 1990s, respectively. Recently, the non-dominant genotypes have been implicated in numerous JE outbreaks, posing significant public health challenges. This study conducted a comprehensive phylogenetic analysis of 126 JEVs covering five genotypes from 1938 to 2025 globally. Notably, it is the first study to conduct a time to most recent common ancestor (tMRCA) analysis of JEV GII. The findings indicate the common ancestor of JEV emerged approximately 1081 years ago, followed by the sequential evolution of GV, GIII, GI, GII, and GIV, which are estimated to have originated approximately 237, 184, 138, 130, and 113 years ago, respectively. The overall evolutionary rate of JEV was 3.3 × 10^-4 substitutions/site/year, with genotype specific rates as follows: GI at 7.3 × 10^-4, GII at 8.2 × 10^-4, GIII at 4.4 × 10^-5, GIV at 1.7 × 10^-3, and GV at 2.2 × 10^-3. Significant differences were found between the E protein of recent human-derived JEV strains across five genotypes and the vaccine strain. This aligns with evidence of the current JE vaccine's reduced efficacy against new strains, underscoring the urgent need for broadly protective JE vaccines.