Origin and evolution of Japanese encephalitis virus in southeast Asia

Abstract

Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.

FIG. 1.

Phylogenetic trees of JEV. (A) Neighbor-joining phylogeny of complete JEV genomes, with a representative strain from other viruses in the JEV serogroup (KUN, Kunjin; MVE, Murray Valley encephalitis; SLE, St. Louis encephalitis; WN, West Nile), outgrouped by using Dengue-2 strain New Guinea C. (B) Neighbor-joining phylogeny of envelope genes, outgrouped by using MVE strain 1-51. Indonesian isolates are boxed. The tree was initially constructed by using more than 200 isolates from all geographical areas, but for clarity only a representative isolate of each genotype from each geographical area is shown. Isolates sequenced in this paper are shown with arrows. Genotypes are given on the right of each tree (5, 38). Bootstrap support values, given as a percentage of 1,000 replicates, are shown.

FIG. 2.

The geographical distribution and spread of JEV genotypes. Countries are grouped into geographical regions: A, Indonesia (excluding New Guinea) and Malaysia; B, Australia and New Guinea; C, Taiwan and the Philippines; D, Thailand, Cambodia, and Vietnam; E, Japan, Korea, and China; F, India, Sri Lanka, and Nepal. Region A contains all genotypes of JEV, including the oldest. The newer genotypes (I, II, and III) have subsequently spread to other geographical areas.

FIG. 3.

The variable region of JEV genotype IV capsid (C) protein. Genotype IV strain JKT6468 was aligned with JEV strains K94P05 (genotype I), FU (II), JaOArS982 (III), Muar (V), and MVE 1-51. The variable region of 20 amino acids just proximal to the carboxy terminus of genotype IV viruses is shown here. Amino acids are compared with those of JaOArS982; dots indicate conserved residues, dashes indicate gaps. A second genotype IV strain (Indonesian strain JKT7003) was also sequenced for this region and had identical sequence to JKT6468. Although this region of the C protein is markedly different for genotype IV, it still includes a hydrophobic transmembrane domain (underlined). Shaded regions indicate the predicted signalase/protease cleavage motifs. Boxes indicate the predicted N-linked glycosylation sites.

FIG. 4.

JEV envelope (E) protein. (A) Predicted three-dimensional model of the E protein of JEV genotype IV strain JKT6468, derived from the crystal structure of TBEV (27), showing E38 in domain I (the hinge domain), and E327 on the exposed surface of domain III. (E399 is in the stem region from domain III into the membrane anchor; not shown). (B) The critical change at residue 327 (in the putative receptor binding region of domain III) from the aliphatic amino acid leucine in genotype IV strains to the hydroxyl-containing serine found in newer genotypes.