Genotypic and Phenotypic Determinants of Japanese Encephalitis Virus Geographical Expansion

Japanese encephalitis virus (JEV) consists of at least four geographically and epidemiologically distinct genotypes (GI-IV). Genotypes II and IV have been isolated in tropical Asia only, and GI has recently replaced GIII as the dominant JEV genotype throughout Asia. Therefore, the objective of this dissertation was to elucidate genotypic and phenotypic determinants of JEV geographical expansion by relating differences in the molecular epidemiology of virus isolates to the phenotypic properties of the isolates through in vitro and in vivo investigations. Phylogeographic analysis revealed that GI consisted of two genetically and geographically distinct clusters, GI-a (tropical Asia only)and GI-b (primarily temperate Asia; emergent cluster). Further, it was demonstrated that both GIII and GI-b are temperate genotypes, suggesting that the spread and establishment of GI-b throughout Asia may have been due to its ability to efficiently overwinter in temperate Asia. Phylodynamic analysis indicated that a decade prior to the genotype displacement the relative genetic diversity of GI had surpassed that of GIII, thereby conferring a selective advantage to GI. No differences in viral multiplication were observed among viral isolates representative of GI-IV of JEV in avian cells; however the GI-b isolate had significantly higher infectious titers in C6/36 Aedes albopictus cells (mosquito larvae derived cell line lacking a functional RNA interference response) from 24-48 hours post infection compared to the other viral isolates. In nature, an increased viral multiplication efficiency of GI-b viruses in mosquitoes may have resulted in a shortened extrinsic incubation period leading to an increased number of GI enzootic transmission cycles and the subsequent displacement of GIII. The blackbird model of JEV viremia revealed that 100.0% of blackbirds developed detectable viremias following inoculation with the GI-a and GI-b isolates, while only 66.7% of blackbirds developed viremias following inoculation with the GII and GIV isolates. These results suggest that GI-b may have emerged and established itself throughout Asia due to an increased multiplicative ability in avians, and genotypes II and IV may be limited to tropical Asia due to a decreased multiplicative ability in avians.