Genetic Characterization of Japanese Encephalitis Virus Genotype 5 Isolated from Patient, South Korea, 2015

Abstract

J apanese encephalitis is caused by Japanese encephalitis virus (JEV), a mosquitoborne virus of the family Flaviviridae, genus Flavivirus (1). The JEV genome is composed of a single-stranded, positive-sense RNA of ≈11 kb with a single open reading frame (ORF) encoding a polyprotein. The polyprotein is processed into 3 structural proteins, capsid, membrane, and envelope (E), and 7 nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (2).JEV is distributed in temperate and tropical areas of eastern and Southeast Asia. In 2010, JEV genotype 1 was the predominant virus circulating. However, genotype 5 was also identified in mosquitoes in South Korea (3). Since that time, JEV genotype 5 has been detected in mosquitoes in many areas of South Korea (4). We report isolation of JEV genotype 5 virus from patient specimens and differences in sequences among other JEV strains (genotypes 1-5). The StudyWe isolated JEV (strain K15P38) from samples of a 27-year-old woman who came to a hospital in Kyeonggi-do, South Korea, on November 8, 2015.The patient had mild symptoms, such as fever, headache, apathy, and nausea. The patient recovered. We obtained documentation from the hospital that she had been vaccinated against Japanese encephalitis. Cerebrospinal fluid (CSF) and serum samples were obtained during the acute and convalescent phases.We detected JEV IgM in serum and CSF samples by using an ELISA (Inbios, https://inbios.com) for convalescent-phase samples, but not acute-phase samples. We isolated virus by inoculating the convalescent-phase CSF sample onto BHK-21 cells. After a cytopathic effect was observed, we confirmed presence of virus by using a quantitative real-time PCR. We performed whole-genome sequence analysis of the virus by using virus genome extracted from 5 passaged culture supernatants and QIAamp Viral RNA Mini Kit (QIAGEN, https://www.qiagen.com).We performed next-generation sequencing for full-length genes by using the Illumina (https://www. illumina.com) and confirmed gaps from next-generation sequencing by using Sanger sequencing. We assembled nucleotide sequences by using the SeqMan program in DNASTAR software version 5.06 (https://www. dnastar.com). We then conducted molecular phylogenetic analysis of ORF nucleotide sequences with 30 previously reported JEV strains by using MEGA 6.0 software (https://www.megasoftware.net) and the maximum-likelihood method (5) and calculated timescale phylogenies by using BEAST version 2.6.0 software (6). We deposited the polyprotein genome sequence of strain K15P38 in GenBank (accession no. MK541529).We compared the entire ORF sequences of K15P38 virus with previously reported strains of JEV genotypes 1-5. Phylogenetic analysis showed that K15P38 belonged to JEV genotype 5 by (Figures 1,2,

Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes

“…JEV GV was isolated from Cx . tritaeniorhynchus mosquitoes in China in 2009 [ 9 ] and from human specimens in South Korea in 2015 [ 11 ]. It was also detected in Cx .…”

“…The other three genotypes (GII, GIV, and GV), on the other hand, seem to be confined to the Indo-Malay region [ 7 , 8 ]. Although JEV GV used to be a rare lineage with relatively limited distribution, it is now considered to be re-emerging following recent detection in mosquitoes in China and South Korea [ 9 , 10 ] and in a JE-vaccinated human patient in South Korea [ 11 ]. Accordingly, as well as the two dominant genotypes (GI and GIII), JEV GV was included in this study because it is considered a rising threat in Japan.…”

Identification of Japanese Encephalitis Virus Genotype V and Other Mosquito-borne Viruses in Camp Humphreys, Republic of Korea, using Metagenomic Analysis

“…Initially, the endemic JEV genotype identified in the ROK was Genotype III (GIII) until ∼1990 when it shifted to Genotype I (GI) and then most recently to Genotype V (GV), which was first identified in 2010. Prior to our study, only four full JEV GV genomes were available in GenBank for sequence comparison, collected in 1952 (two nearly identical sequences reported), 2009, and 2015 ( Figure 4B ) (23, 25, 43). We utilized targeted sequencing of our JEV positive pools to assemble three full JEV genomes and we confirmed that they belonged to GV using full CDS phylogenomics ( Figure 4C ).…”

“…Within the Republic of Korea (ROK), JEV GV was first detected in Culex bitaeniorhynchus mosquitoes, collected from Daeseongdong (a village in northern Gyeonggi province located in the demilitarized zone) in 2010, with subsequent detection in Culex orientalis and Culex pipiens collected in the Gangwon and Gyeonnggi provinces (9, 24). Recently, JEV GV has been reported in clinical cases in the ROK (25).…”

Amino Acid at Position 166 of NS2A in Japanese Encephalitis Virus (JEV) Is Associated with In Vitro Growth Characteristics of JEV

“…In Korea, GI, GIII, and GV JEV strains have been endemic in recent years, but GV is currently the main genotypic strain circulating in Korea [ 12 , 13 ]. Furthermore, GV JEV was isolated from a JE patient in 2015 [ 14 ].…”