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,