Dynamics of the emergence and establishment of a newly dominant genotype of Japanese encephalitis virus throughout Asia

In recent years, genotype I (GI) of Japanese encephalitis virus (JEV) has displaced genotype III (GIII) as the dominant virus genotype throughout Asia. In this study, the largest collection of GIII and GI envelope gene-derived viral sequences assembled to date were used to reconstruct the spatiotemporal chronology of the genotype displacement throughout Asia, and to determine the evolutionary and epidemiological dynamics underlying this significant event. GI consists of two clades, GI-a and GI-b with the latter associated with displacement of GIII as the dominant JEV genotype throughout Asia in the 1990s. Phylogeographic analysis indicated that GI-a diverged in Thailand or Cambodia and has remained confined to tropical Asia, whereas GI-b diverged in Vietnam and then dispersed northwards to China, where it was subsequently dispersed to Japan, Korea and Taiwan. Molecular adaptation was detected by > 1 method at one site (15) and co-evolution was detected at two pairs of sites (89-360 and 129-141) within the GI E gene protein alignment. Viral multiplication and temperature sensitivity analyses in avian and mosquito cells revealed that the JE-91 GI-b isolate had significantly higher infectivity titers in mosquito cells from 24-48 hours post infection compared to the GI-a and GIII isolates. If the JE-91 isolate is indeed representative of GI-b, an increased multiplicative ability of GI-b viruses compared to GIII viruses early in mosquito infection may have resulted in a shortened extrinsic incubation period that led to an increased number of GI enzootic transmission cycles and the subsequent displacement of GIII.IMPORTANCE Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, represents the most significant etiology of childhood viral neurological infection in Asia. Despite the existence of effective vaccines, JEV is responsible for an estimated 68,000 human cases and a reported 10,000 to 15,000 deaths annually. Phylogenetic studies divide JEV into five geographically and epidemiologically distinct genotypes (GI-V). GIII has been the source of numerous JE epidemics throughout history and was the most frequently isolated genotype throughout most of Asia from 1935 until the 1990s. In recent years, GI has displaced GIII as the most frequently isolated virus genotype. To date, the mechanism of this genotype replacement has remained unknown. In this study we have identified genetic determinants underlying the genotype displacement as it unfolded across Asia. JEV provides a paradigm for other flaviviruses, including West Nile, yellow fever and dengue viruses, and the critical role of the selective advantages in the mosquito vector.