First Molecular Detection of Japanese Encephalitis Virus Genotype I in Culex Mosquitoes from Assam, Northeast India

Japanese encephalitis (JE) continues to pose a major public health threat in northeast India, particularly in Assam, where recurring outbreaks highlight the need for strengthened vector surveillance. In May 2025, after JE infection was confirmed in pigs in Kamrup District, an entomological investigation was conducted to assess mosquito vector abundance and detect circulating Japanese encephalitis virus (JEV). Adult mosquitoes and larvae were collected from JE-affected and high-risk areas, and the standard WHO method was used to calculate mosquito density, which was 143 per-man-hour density. A total of 858 mosquitoes were collected, of which 521 (60.7%) were adult female Culex spp. Culex tritaeniorhynchus was the predominant species followed by Culex quinquefasciatus and Culex gelidus. Twenty mosquito pools were tested for JEV using reverse transcription polymerase chain reaction targeting the C-prM gene; three pools of Cx. tritaeniorhynchus tested positive, corresponding to a minimum infection rate of 5.75. Sanger sequencing and phylogenetic analysis confirmed all positive samples as genotype I (GI), showing high similarity to globally circulating GI strains. Spatial mapping revealed that JEV-positive pools were clustered within 2 kilometers of the index site, mainly around pig farms, wetlands, and paddy fields. This investigation provides the first molecular evidence of JEV GI circulating in mosquito populations in Assam. The detection of GI in the primary vector confirms its circulation in Assam and highlights the need for continued molecular surveillance. These findings underline the urgent need for integrated vector surveillance, targeted control measures, and One Health-based monitoring in Assam.