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

Abstract

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 was used to reconstruct the spatiotemporal chronology of 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 being 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 more than one method at one site (residue 15), and coevolution was detected at two pairs of sites (residues 89 to 360 and 129 to 141) within the GI E gene protein alignment. Viral multiplication and temperature sensitivity analyses in avian and mosquito cells revealed that the GI-b isolate JE-91 had significantly higher infectivity titers in mosquito cells from 24 to 48 h postinfection than did 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 that of 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 divided JEV into five geographically and epidemiologically distinct genotypes (GI to GV). GIII has been the source of numerous JEV 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.

FIG 1

Relative proportions of GIII and GI according to decade of collection.

FIG 2

Bayesian skyline reconstruction for GIII versus GI.

FIG 3

Geographical distribution of the GI sequences included in this study. For each country included in the GI phylogeographic analysis, the estimated date of the MRCA, the dates of virus collection, and the number of viral sequences that were included in this study are indicated. The direction of GI dispersal is also indicated. The direction of GI dispersal, based on pathways between countries with a BF of ≥3 in the BSSVS analysis, is also indicated.

FIG 4

GI Bayesian MCC phylogeny. GI-a and GI-b are represented to the right of the tree. Branch tips correspond to the date of collection of each of the virus isolates. Branch lengths correspond to lengths of time, as measured by the scale located under the tree. Terminal branches are colored according to the sampling location of the taxon at the tip, while internal braches are colored according to the most probable location of their child node. The numbers at the nodes correspond to the phylogeographic analysis data presented in Table 2. Asterisks represent the initial divergence of the MRCA of each of the seven countries included in the GI phylogeographic analysis. Clusters of closely related viruses that were isolated in temperate Asia (Japan, Korea, and China) from year to year are indicated to the right of the phylogeny.

FIG 5

Multiplication kinetics of isolates representative of GI to GIV in avian and mosquito cells. (A) DEF cells incubated at 37°C; (B) DEF cells incubated at 41°C; (C) C6/36 cells incubated at 28°C. The lower limits of detection were 10 PFU/ml for samples taken at 0 to 36 hpi and 100 PFU/ml for samples taken at 48 hpi and onwards. For panel C, error bars represent SD values based on triplicate experiments.