Abstract Introduction: Japanese encephalitis virus (JEV), in the genus Flavivirus, family Flaviviridae, is the leading cause of viral encephalitis in the Asian-Pacific region. JEV has a wide range of vector hosts, including mosquitoes, swine, and wading birds. Culex species, especially Culex tritaeniorhynchus, are the main vectors for JEV transmission. JEV has been classified into five genotypes (G1–G5). JEV G1, G3, and G5 isolates have been found in China. Methods: More than 200,000 mosquitoes collected in 14 provinces in China from 2004 to 2016 were examined for JEV using a TaqMan real-time RT-PCR assay. The JEV envelope (E) gene was sequenced from positive pools. The nucleic acid sequences were analyzed and aligned using ClustalX ver. 2.0. Representative JEV envelope (E) gene sequences were downloaded from GenBank and compared with the newly obtained JEV sequences. MEGA 5.05 was used to generate a phylogenetic tree based on the JEV sequences. Results: More than 200,000 mosquitoes were divided into 3107 pools to test for JEV. Of the pools, 9.04% (281/3107) were positive. The JEV obtained from mosquitoes included three genotypes (G1, G3, and G5). JEV G1 was detected from different provinces during 2004–2016. G1 was the dominant genotype circulating in nature, comprising 98.93% (278/281). JEV G3 was detected in Yunnan and Chongqing Provinces before 2010; JEV G5 was detected only in Tibet. No pools were positive for JEV G2 and G4. C. tritaeniorhynchus was the major mosquito species and the principal vector for transmitting JEV in China. The results of the TaqMan real-time RT-PCR assay and phylogenetic analysis consistently indicated that all 50 newly identified JEV sequences belonged to JEV G1. Conclusion: G1 is the main genotype of JEV circulating in nature in China. Some G3 and G5 were found, but no G2 or G4.
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