Genetic diversity of Japanese encephalitis virus isolates obtained from the Indonesian archipelago between 1974 and 1987

Abstract

Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate of the Indonesia/Malaysia region, together with its plethora of distinct fauna and flora, may have driven the emergence and evolution of JEV. This is consistent with the extensive genetic diversity seen among the JEV isolates observed in this study, and further substantiates the hypothesis that JEV originated in the Indonesia-Malaysia region.

FIG. 1.

Map of the Indonesian archipelago. The islands comprising Indonesia are shown in white, large text indicates the islands where Japanese encephalitis virus (JEV) isolates included in this study were collected, and small text indicates the cities in Java where JEV isolates included in this study were collected. Twenty-seven of the Indonesian isolates were from the island of Java (one from Jakarta, 19 from Kapuk, one from West Java, three from Central Java, one from Bantul, and two from unspecified locations in Java), two were from the island of Bali, three were from the island of Lombok, one was from the island of Flores, and one was from an unspecified location in Indonesia. The map was created using the ArcView GIS version 9.1 (Environmental Research Systems Institute 2004) from geographic boundary files downloaded from the DIVA-GIS, Geodatabase (DIVA-GIS, 2011). (Color image available online at www.liebertpub.com/vbz).

FIG. 2.

Neighbor-joining phylogeny based on nucleotide sequence information derived from the E gene of the Japanese encephalitis virus (JEV) isolates. The tree was rooted using the MVE-1-51 isolate of Murray Valley encephalitis virus, which is a member of the Japanese encephalitis serogroup, but has been removed to allow for better visualization of branch lengths. Horizontal branch lengths are proportional to the genetic distance between strains, and the scale at the lower-left of the tree indicates the number of nucleotide substitutions per site. Genotype (GI–IV) is represented to the right of the tree. Bootstrap percentages based on 1000 replicates are indicated to the lower-left of the genotype-defining nodes within the phylogeny, as well as additional selected nodes. The Indonesian isolates sequenced in this study are indicated by asterisks.

FIG. 3.

The location of important amino acids sites mapped onto the three-dimensional E protein structure of Japanese encephalitis virus (JEV) (PBD ID: 3P54, MMDB ID: 87213): (A) Node-defining amino acid substitutions specific to the Indonesian viruses sequenced in this study. (B) Sites identified by the directional evolution in protein sequences (DEP)S analysis to exhibit evidence of directional evolution. (C) Sites detected by the Spidermonkey algorithm to display evidence of co-evolution. Domain I is indicated in red, domain II in yellow, and domain III in blue. The image was created using MacPyMOL version 1.3. (Color image available online at www.liebertpub.com/vbz).