Detection of Japanese encephalitis virus genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea

Abstract

Japanese encephalitis virus (JEV) causes significant viral encephalitis and is distributed throughout the Asian countries. The virus is known to be transmitted by Culex tritaeniorhynchus, which mainly breeds in rice paddies in Korea. In this study, we investigated the presence of other mosquito species that can transmit JEV as a second or regional vector. We selected five cities where patients have experienced JE in the last 5 years as mosquito-collecting locations and subdivided them into four collection sites according to the mosquito habitats (cowshed, downtown area, forest, and swamp). Mosquitoes were caught using the BG-Sentinel trap, CDC black-light trap, Fay-Prince trap, and Gravid trap. A total of 993 pools from 22,774 mosquitoes were prepared according to their species, collection date, and site. We performed a SYBR Green 1-based real-time RT-PCR assay to detect JEV from the mosquito pools. A total of six JEV-positive pools were detected from Culex orientalis and Culex pipiens caught in the Gangwon-do and Gyeonngi-do provinces. All the detected JEVs were revealed as genotype V by phylogenetic analysis of the envelope gene. Our findings confirm that a new genotype of JEV was introduced in Korea and suggest that two mosquito species may play a role in JEV transmission.

Figure 1. Locations of mosquito collection in South Korea.

Mosquitoes were caught in five cities (four sites per city) once a month from May through October 2012.

Figure 2. Phylogenetic analysis of Japanese encephalitis virus (JEV) based on the complete envelope gene (1,500 nt) from 42 strains representing each genotypes and countries.

The Maximum-Likelihood tree was constructed with a substitution model of Tamura-Nei plus gamma distribution using MEGA software 6.06. West Nile virus (WNV, B956 strain, NC_001563) was used as the outgroup in the tree. Branch reliability is indicated by the percentage of bootstrap values at each node (1,000 replications). The scale bar indicates the number of base substitutions per site. JEVs detected in this study were marked with a closed circle. The left side of the tree is omitted for ease of understanding. In a small rectangle, the topology of genotype V sequences in the original tree is presented. The branch length between K12AS 1154 and its ancestral node is zero and is edited to make the ancestor-descendant relationship clear.