First evidence of the presence of genotype-1 of Japanese encephalitis virus in Culex gelidus in Indonesia
Japanese encephalitis has become a public health threat in Indonesia. Three genotypes have been recorded in Indonesia, i.e. genotype II (GII), genotype III (GIII) and genotype IV (GIV). Genotype I (GI) and genotype V (GV) have never been reported in Indonesia.
A Japanese encephalitis virus (JEV) belonging to the genotype I-a (GI-a) has been isolated for the first time from a Culex gelidus mosquito in the Province of Jambi, Indonesia. This virus is related to a 1983 isolate from Thailand whereas the infected Cx. gelidus mosquito belonged to a Chinese haplotype.
Surveillance of JEV and mosquito dissemination is recommended.
KeywordsJapanese encephalitis Genotype I Indonesia Culex gelidus
Japanese encephalitis virus
cytochrome c oxidase subunit 1
Murray Valley encephalitis virus
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has become a public health threat in Asia, including Indonesia. JEV is transmitted to humans through mosquito bites, especially of Culex species, from amplifier animals such as pigs. JEV can cause severe central nervous system disorders with high mortality or permanent neurological sequelae .
In Indonesia, JEV was first isolated from mosquitoes in West Java in 1972. Since then, encephalitis cases have been reported in several hospitals and currently Japanese encephalitis (JE) has become widespread and endemic across 32 out of 34 Indonesian provinces . JEV originated from the IndoMalayan region and further evolved into five genotypes. Until now, only three genotypes have been recorded in Indonesia, i.e. genotype II (GII), genotype III (GIII) and genotype IV (GIV). Furthermore, GIV has only been described in mosquitoes in Indonesia . Although JEV originated from the Indo-Malaysia region about 1695 years ago, genotype I (GI) most likely originated in Thailand for clade GI-a and Vietnam for clade GI-b about 193 years ago and has never been reported in Indonesia [3, 4]. Genotype I is associated with human encephalitis in China, Japan, India, Korea, Taiwan, Thailand and Vietnam . GI JEV is an epidemic genotype with equal virulence as GIII JEV, the genotype most frequently associated with outbreaks in Asia .
The amplification products were then purified using Illustra ExoProSTar (GE Healthcare Life Sciences, Tokyo, Japan). Sequencing of the amplified gene E was performed using the primers JEV-Ef and JEV-Er mentioned above and ABI BigDye terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems, Austin, USA). Prior to sequencing, a PCR product purification step was performed using BigDye Xterminator Purification Kit (Applied Biosystems). Sequence data were obtained through the Sanger method using an automatic DNA sequencer (Applied Biosystems 3500 Genetic Analyzer) and analyzed using the Sequencing Analysis 6 program (Applied Biosystems).
The phylogenetic tree was built using the maximum likelihood method with Tamura Nei (TN93) as evolutionary model. Node bootstraps were calculated with 2000 replicates. Sequences were compared to 16 JEV reference sequences from GenBank comprising 6 GI-b, 2 GI-a, 2 GII, 3 GIII, 2 GIV, 1 GV and 2 MVEV (Murray Valley encephalitis virus) sequences used as outgroups. The mosquito in which the JEV was detected was genotyped using the cytochrome c oxidase subunit 1 gene (cox1) as a target. The cox1 gene was amplified using the primers CIN2087 (5'-AAT TTC GGT CAG TTA ATA ATA TAG-3') and TYJ-1460 (5'-TAC AAT TTA TCG CCT AAA CTT CAG CC-3') as previously described .
Results and discussion
Culex mosquitoes captured in the Province of Jambi, Indonesia
No. of pools
No. of samples
To our knowledge, this is the first detection of GI JEV in Indonesia. A replacement of GIII by GI was reported throughout Asia and Australia since 1979 . A similar phenomenon could be underway in Indonesia and should be investigated more thoroughly. The mode of introduction of GI in several countries has not been clearly established but a narrower vector host range and a higher replication capacity of GI in mosquitoes has been described . The cox1 haplotype of the Cx. gelidus mosquito infected with JE/mosq/Jambi107/2017 (GenBank: MK045308) was found to be very close to a haplotype previously described in China (GenBank: MF179173). Blast results for the two best hits were as follows: GenBank: MF179173, maximum score 861, total score 861, total coverage 100%, E value 0.0, identity 97%; GenBank: MF179172, maximum score 856, total score 856, total coverage 100%, E value 0.0, identity 97%. Culex gelidus is a good vector of JEV with an invasive capacity and a potential for being transported over long distance by boats, planes or road transportation .
The role of specific mosquito populations in the introduction and dissemination of GI JEV through commercial routes should be investigated. GI, which is currently replacing GIII in Asia, could not be detected in cerebrospinal fluid by JEV-specific IgM antibodies raised against GIII JEV . There is thus a risk of misdiagnosis in the presence of GI. Furthermore, all vaccines currently available against JEV are derived from GIII JEV and several studies have reported human confirmed cases with GI JEV infection in areas where effective JEV vaccination programs exist [11, 12]. There is thus, in addition to misdiagnosis, a risk of lack of efficient protection associated with the extension of GI. Further studies and strengthened JE surveillance should be implemented to assess the distribution of GI JEV in Indonesia and health authorities must be alerted in order to address potential risks to public health.
The authors express their gratitude to the head of the National Institute of Health Research and Development, the Ministry of Health of Indonesia for permission to do the analyses. The authors would also like to thank the people in the study areas for cooperating during the entomological surveys, all field mosquito collectors from the Rikhus Vektora project in Jambi, and the experts and technical team of the Rikhus Vektora project for fruitful discussions.
The research was supported by the Institute for Vector and Reservoir Control Research and Development, National Institute of Health Research and Development, Ministry of Health Indonesia under the project Rikhus Vektora 2017. SM and RF were supported by the Université de Montpellier, IRD and CIRAD, Montpellier, France.
Availability of data and materials
Data supporting the conclusions of this article are included within the article. Raw data are available from corresponding author upon reasonable request. Sequences are deposited in the GenBank database under the accession numbers MK032889 and MK045308.
TAG, JW, TTS and LS conceived and designed the experiments. TAG, MTP, LS. DP, SRS, YT, TBTS and DP performed the experiments. TAG, MTP, SM and RF analyzed the data. TAG, TTS, SM and RF wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The ethical clearance for primary data was obtained from the authorities of the Ethics Commitee of the National Institute of Health Reserach and Development, Ministry of Health of Indonesia (number LB.02.01/5.2/KE.020/2017).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 2.Soedarmo SP, Wuryadi S, Suroso T. Japanese encephalitis in Indonesia. In: Regional workshop on control strategies for Japanese encephalitis. Nonthaburi, Thailand, 4–6 October; 1994.Google Scholar
- 10.Whelan P, Hayes G, Tucker G, Carter J, Haigh B, Whelan P, et al. The detection of exotic mosquitoes in the Northern Territory of Australia. Arbovirus Res Aust. 2001;8:395–404.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.