Complete Genome Sequences of Human Japanese Encephalitis Virus Genotype V Isolates in Korea Reveal Genotype-Specific Amino Acid Signatures

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus causing severe neurological disease across Asia, and genotype V (GV) is now predominant in Korea. Despite frequent detection of GV in mosquitoes, human-derived complete genome data remain scarce. To elucidate the molecular and antigenic characteristics of human GV infections, cerebrospinal fluid samples from unvaccinated patients positive for JEV RNA during 2018–2023 were subjected to virus isolation in LLC-MK2 cells (rhesus monkey kidney-derived epithelial cell line). Three human GV isolates (K18P80, K23P84, K23P88) were successfully obtained and their complete open reading frames (~10.3 kb) sequenced. Phylogenetic analysis with representative JEV strains (GI–GV) revealed that these isolates form a distinct lineage, clustering into two domestic clades (Clade I and II), suggesting endemic circulation and local evolution in Korea. Sequence identities with GIII-based vaccine strains were low (79% nucleotide, 91.1% amino acid), with notable divergence in nonstructural regions. Three consistent E protein substitutions (Q52E, S156T, D292E) near antigenic epitopes indicate possible immune escape. Additional clade-defining substitutions in NS3 (L31F) and NS5 (K269R, M330I) were shared with mosquito isolates, supporting human–vector molecular continuity. These findings provide fundamental genomic evidence of human JEV GV in Korea and highlight the need for genotype-specific surveillance and next-generation vaccine evaluation.