Isolation and characterization of Japanese encephalitis virus genotype I from pig provides evidence for the presence of the virus in nasal secretion and co-circulation of JEV genotypes in Assam, India

Background Japanese encephalitis (JE) is a leading cause of viral encephalitis, among children, in many Asian countries despite the availability of effective vaccines. There are five JE virus genotypes (GI through GV), with GIII being the most prevalent. However, in the past three decades GI has emerged as the dominant genotype across several Asian countries, while the reappearance of GV is a concern due to the reduced cross-neutralization offered by existing GIII-based vaccines. Although both GI and GIII have been reported to co-circulate in India, all previous JEV isolations from pigs have been of the GIII. Objective The objective of the study was to elucidate the JEV genotype diversity among pigs in Assam through molecular and virological investigation. Methods We collected blood, serum and nasal swab samples from apparently healthy pigs as a part of routine disease surveillance in pigs of Kamrup (Rural) district, Assam, India. The samples were processed using standard molecular biology (qRT-PCR, Gene Sequencing, Phylogenetic analysis) and virological techniques (Virus isolation, immunofluorescence, plaque assay) for JE virus detection, isolation and characterization. Results In this study, we report the first isolation and characterization of a JEV GI from a nasal swab of a naturally infected pig from Assam, India and the isolate was designated as JEV/Pig/Assam/NIVEDI-1/2025 (GI). The identity of the JEV isolate was confirmed by RT-qPCR, phylogeny based on 5′UTR–prM region, full-length envelope protein gene, and immunofluorescence assay. The isolate reached a peak titer of 10 6.5 TCID 50 /mL at 72 h post-infection in Porcine stable kidney cells and produced smaller plaques (1.88 ± 0.56 mm) than the reference GIII strain (2.68 ± 0.48 mm) ( p 0.01). Conclusions The findings underscore that JEV GI is circulating in Assam and there is need for strengthened JEV surveillance in swine to monitor genotype shifts, understand viral evolution, and generate field isolates critical for vaccine evaluation and preparedness against emerging JEV genotypes. The study also demonstrates the feasibility of using nasal swabs for virus detection and isolation thereby providing evidence for the presence of JEV in nasal secretion of naturally infected pigs.