This study was approved by the Committee for Prevention and Supervision of Experiments on Animals (CPCSEA) with the approval number V-11011(13)/7/2025-CPCSEA-DADF in 2025.

The samples were collected by the trained veterinarian following the national animal ethical guidelines of the Government of India. Informed verbal consent was obtained from pig farmers before the sample collection.

During May 2025, following notification by the local public health department regarding suspected JE activity in Goshaihaat village, Kamrup (Rural) district, Assam, India, ICAR-National Research Centre on Pig (ICAR-NRC on Pig), Guwahati and ICAR-National Institute of Epidemiology and Disease Informatics (ICAR-NIVEDI), Bengaluru conducted JEV surveillance among pigs in the village. The villagers maintained Large White Yorkshire and Duroc crossbred pigs and all the animals were clinically healthy at the time of sampling. In the first-round, blood samples from pigs (n=34) of 17 households were screened for JEV infection between 21–23 May 2025. Of the samples tested, pigs from two farms (hereafter mentioned as Farm A and Farm B were found positive for JEV by RT-PCR (Table 1, Figure 1). Based on these results second round of samples (n=8) were collected for testing from those two JEV positive farms on 29 May 2025. Whole blood, serum, and nasal swab samples were collected aseptically from pigs following standard biosafety protocols. The farm- and animal-level details of JEV RT-PCR positive farms are provided in Table 2.

In the third round of sample collection, 30 pig serum samples were collected from the same village during 9–11 June 2025 including the Farm A and B. The samples collected on 21–23 May 2025 and June 2025 were screened at ICAR-NRC on Pig and those collected on 29 May 2025 were screened at ICAR-NIVEDI.

Japanese encephalitis virus strain JEV/eq/India/H225/2009 (H225) isolated from horse and porcine stable kidney (PS) cells were kind gift from Virology Laboratory, ICAR-National Research Centre on Equines, Hisar. JEV SA14-14–2 vaccine was obtained from Department of Health Services, Government of Karnataka. Vero and PS cell lines were maintained in Eagle’s minimal essential medium (MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin-B. The cultures were incubated at 37 °C in a humidified atmosphere with 5% CO₂ and routinely monitored for cell viability and contamination.

Total RNA was extracted from whole blood, nasal swabs and cell culture supernatant using NucleoSpin RNA Blood Mini kit (Macherey-Nagel, Germany), and QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) following manufacturer’s protocols.

The cDNA was synthesized from 10 µl total RNA using random hexamers and RevertAid™ M-MuLV Reverse Transcriptase (Thermo Scientific, USA), as per the manufacturer’s instructions. Detection of JEV was carried out using both conventional and quantitative reverse transcription PCR (RT-PCR) assays. A two-step conventional RT-PCR targeting the NS3 gene, 5′ UTR–prM region and the Envelope gene regions, and a one-step RT-qPCR targeting the NS2A gene were performed as described previously (Tanaka, 1993; Yeh et al., 2010; Jeong et al., 2011; Bharucha et al., 2018). The one-step RT-qPCR was performed in a 20 µL reaction volume using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, USA). Each reaction contained 600 nM of forward and reverse primers, 300 nM of probe, and 6.2 µL of extracted RNA template. The amplification protocol consisted of reverse transcription followed by 40 amplification cycles.

RNA extracted from cell culture supernatant of PS cell line infected with JEV SA 14-14–2 strain was used as positive control, while nuclease-free water as negative control in each RT-PCR run. Amplicons obtained from the conventional RT-PCR were visualized by agarose gel electrophoresis, purified using standard gel extraction procedures, and subjected to Sanger sequencing for confirmation of JEV identity.

Samples that tested positive for JEV by conventional RT-PCR were subjected to virus isolation in cell culture. Briefly, 500 µL of whole blood or nasal swab homogenate, filtered through a 0.22 µm syringe filter, was inoculated onto a sub-confluent monolayer of Vero cells in a T-25 flask. Virus adsorption was allowed for 1 h at 37 °C with intermittent gentle rocking. After adsorption, the inoculum was removed, and the cell monolayer was washed with phosphate-buffered saline (PBS, pH 7.4) to remove unbound virus. Subsequently, 5 mL of Eagle’s Minimum Essential Medium (EMEM) supplemented with 2% fetal bovine serum (FBS) was added, and cultures were incubated at 37 °C in a 5% CO₂ atmosphere. Infected flasks were monitored daily for cytopathic effects (CPE), characterized by cell rounding, detachment, and aggregation. Each sample underwent up to four blind passages before being declared negative for virus isolation. When CPE involved approximately 70% of the cell monolayer, the culture flask was frozen at −80 °C and subjected to three freeze–thaw cycles to release intracellular virus. The culture supernatant was clarified by centrifugation at 200 × g for 10 minutes at 4 °C, aliquoted, and stored at −80 °C for further analysis. The presence of JEV in the culture supernatant was confirmed by both conventional RT-PCR, RT-qPCR, gene sequencing, and indirect immunofluorescence assay (IFA).

The indirect immunofluorescence assay (IFA) was performed using PS cells infected with the fifth passage of the JEV isolate to confirm viral antigen expression. Briefly, a confluent monolayer of PS cells in a 25 cm² culture flask was infected with the JEV isolate and incubated at 37 °C for 48 h. Following incubation, the cell monolayer was fixed with 2 mL of ice-cold 80% acetone for 10 min at 4 °C and air-dried. The fixed monolayer was rinsed with 3 mL of phosphate-buffered saline (PBS, pH 7.4) for 5 min at room temperature.

Blocking was carried out using 2 mL of blocking buffer (PBS containing 4% bovine serum albumin [BSA] and 0.05% Tween-20) for 1 h at room temperature, followed by permeabilization with 2 mL of 0.2% Triton X-100 in PBS for 10 min. The cells were then washed three times with 5 mL of washing buffer (PBS containing 2% BSA and 0.01% Tween-20). The JEV NS4B polyclonal antibody (PA5-32211, Invitrogen, USA), diluted 1:2000 in blocking buffer was added (3 mL) and incubated for 1 h at room temperature. After washing three times with washing buffer, the cells were incubated with goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody conjugated with Alexa Fluor™ 488 (A-11008, Invitrogen, USA) diluted 1:1000 in blocking buffer (3 mL) for 1 h at room temperature in the dark. The monolayer was washed thrice with washing buffer, and 1 mL of mounting medium (90% glycerol in PBS, 0.1 M phosphate buffer) was added. Fluorescence was visualized under a fluorescence microscope. A mock-infected flask served as a negative control, and a flask infected with the JEV SA 14-14–2 vaccine strain served as a positive control.

The in-vitro growth kinetics of the JEV isolate (passage 5) was assessed to determine its replication pattern in PS cells. Multiple 25 cm² flasks containing sub-confluent PS cell monolayers were infected with the JEV isolate at a multiplicity of infection (MOI) of 0.01. The cells were maintained at 37 °C in a humidified incubator with 5% CO₂. At 24 h intervals post-infection (0, 24, 48, 72, 96, and 120 h), one flask was frozen at −80 °C. Each flask underwent three freeze–thaw cycles to release intracellular virions, and the culture supernatant was clarified by centrifugation at 200 × g for 10 minutes at 4 °C. The clarified supernatants were aliquoted and stored at −80 °C till further processing. Viral RNA was extracted from supernatant at each time-point sample and subjected to RT-qPCR. Additionally, the clarified supernatants were titrated in 96-well tissue culture plates twice using the Reed–Muench method (Reed and Muench, 1938), and the viral titers were expressed as 50% tissue culture infectious dose per milliliter (TCID₅₀/mL).

The plaque morphology and size of the isolated JEV were compared with those of the reference strain JEV/eq/India/H225/2009 (H225). For the plaque assay, each well of a 6 well plate was seeded with 4 × 10⁵ PS cells and incubated to achieve approximately 90% confluency. The monolayers were infected with 100 µL of an appropriate dilution of either the field JEV isolate (passage 6) or the reference H225 strain (passage 14). After 1 h of virus adsorption at 37 °C, the inoculum was removed, and the wells were washed with PBS. The cell monolayers were then overlaid with a mixture of 2× MEM containing 2% FBS, antibiotics, and 0.6% agarose (yielding a final agarose concentration of 0.3%). The plates were incubated at 37 °C with 5% CO₂ for 96 h. At the end of incubation, the cells were fixed with 10% formal saline for 1 h and stained with 1% crystal violet for 15 min. After rinsing with water and airdrying, plaque morphology and size were recorded. The diameters of 20 randomly selected plaques were measured independently by three observers, and the mean plaque size was calculated. The plaque assay was repeated thrice, and the values were expressed as Mean ± SD.

The template cDNA was amplified using the primers targeting the nucleotide sequence of the 5′ UTR–prM region (505 bp) (Jeong et al., 2011) (fourth cell passage) and full-length envelope gene (1500 bp) (sixth cell passage) (Yun et al., 2010). The PCR amplicons were purified using gel purification and sanger sequencing was performed from a commercial nucleotide sequencing service provider. The sequences were aligned with corresponding regions of JEV reference sequences representing different genotypes (GI–GV) from India and other countries retrieved from the GenBank database. Nucleotide and amino acid similarity matrix was constructed in Bioedit software. Multiple sequence alignment was performed using the MUSCLE algorithm implemented in MEGA 11 software (11.0.13) (Tamura et al., 2021). The phylogenetic tree was constructed using the Maximum Likelihood (ML) method with 1000 bootstrap replicates. The Kimura 2 parameter substitution model with a discrete Gamma distribution (+G) (for 5′ UTR–prM region) and Tamura-Nei model with discrete Gamma distributed with invariant sites (G+I) (for Envelope gene) was applied to account for rate variation among sites (Kimura, 1980). The tree was rooted using the Murray Valley encephalitis virus (GenBank accession number NC_000943) as the outgroup. The robustness of clades was assessed by bootstrap support values, and genotypic clustering was interpreted based on established JEV genotype references.

The serum samples collected during 21–23 May 2025, were subjected to enzyme linked immune-sorbent assay at ICAR-NRC on Pig, Guwahati and those collected on 29 May 2025 were subjected to microneutralization test at ICAR-NIVEDI, Bengaluru. The microneutralization test (MNT) was performed on pig serum samples to detect neutralizing antibodies against JEV, following the protocol described previously (Gulati et al., 2011). Serum samples were heat inactivated at 56 °C for 30 minutes and serially diluted in 2% MEM in 50 µL volumes, starting with a fivefold dilution followed by twofold serial dilutions to yield a dilution series from 1:5 to 1:320. To each diluted serum sample, 50 µL of JEV suspension containing approximately 300 TCID₅₀ was added, and the serum–virus mixtures were incubated at 37 °C for 1 h. The mixtures were then added to preformed PS cell monolayers in 96well plates and incubated at 37 °C with 5% CO₂ for 4 days. Each assay included cell control, serum control, virus back titration control, positive control serum, and negative control serum. The reciprocal of the highest serum dilution that completely inhibited the formation of cytopathic effect (CPE) was recorded as the MNT titer. Samples with titers ≥10 were considered positive for JEV specific neutralizing antibodies.

Of the 34 pigs tested between 21–23 May 2025, seven pigs belonging to two farms (Farm A and Farm B) were found positive by partial envelope gene-based RT-PCR. Based on the preliminary results, the follow-up samples were collected from farm A and B on 29 May 2025 and screened. Of these, blood and nasal swab samples from two pigs (Pig no. 1 and 4) of Farm A were found positive for JEV RNA by NS3 gene-based RT-PCR, while samples from Farm B were negative for JEV (Table 2). The identity of the PCR amplicons was confirmed by gene sequencing.

The RT-PCR–positive blood (n=2) and nasal swab sample (n=1) collected on 29 May 2025 from pig no. 1 and 4 of farm A were subjected to virus isolation in Vero cell culture. The Vero cells inoculated with nasal swab fluid from Pig No. 4 exhibited characteristic CPE including cell rounding and detachment, visible at the second passage (Figure 2). The isolate was successfully maintained through six passages and the strain was designated as JEV/Pig/Assam/NIVEDI-1/2025 (GI). However, two RT-PCR–positive blood samples did not yield viable virus even after four serial passages.

Confirmation of viral presence in all the six cell culture passages of the isolated virus was done by RT-qPCR, with progressive reduction in Ct values across successive passages (Figure 3). The 5′UTR–prM gene (passage 4) and complete envelope gene (passage 6) was sequenced, and the presence of JEV antigen was further confirmed by IFA in passage 5 (Figure 4). The nucleotide sequences were deposited to GenBank with the accession numbers PX489696 (5′ UTR–prM region) and PX489697 (full-length envelope gene).

The PCR amplicon of the 5′UTR–prM (505 bp) and envelope gene (1500 bp) obtained from passage 4 and 6, respectively were sequenced in triplicate, and consensus nucleotide sequence for each gene was derived. Phylogenetic comparison with representative JEV sequences of GI–GV retrieved from GenBank revealed that the isolate belonged to GI. Based on the 5′UTR–prM region the isolate formed a distinct cluster with GI strains reported from Thailand and Cambodia, showing 99% nucleotide identity (Figure 5, Table 3). Comparatively, it shared 95% nucleotide identity with previously reported JEV isolates from Assam, India (Table 3). Similarly, based on envelope gene based phylogenetic analysis, the isolate clustered with GI isolates from Thailand and Cambodia (Figure 5).

The nucleotide and amino acid sequence identity analysis of the envelope gene revealed that the JEV GI isolated in the current study shared 90-92% and 96-98% identity at the nucleotide and amino acid level, respectively with the previous JEV GI sequences from India (Table 4). Further, the nucleotide mutations in envelope gene associated with attenuation of virulence was evaluated by comparing the amino acids of the isolated JEV strain with that of in the JEV SA 14 14 2. It was found that, the JEV GI isolate possessed all the eight critical amino acids found in the neurovirulent JEV strains (Table 5). To the best of our knowledge, this represents the first isolation of a JEV GI strain from the nasal swab of a naturally infected pig in India.

The replication kinetics of JEV/Pig/Assam/NIVEDI-1/2025 (GI) in PS cells demonstrated a progressive increase in virus titer over time. The viral titer was 10^4.45 TCID₅₀/mL at 24 h post infection (p.i.), reached a peak of 10^6.5 TCID₅₀/mL at 72 h p.i., and declined thereafter to 10^3.25 TCID₅₀/mL at 120 h p.i. RT-qPCR analysis of RNA extracted from cell lysates and culture supernatants at sequential time points (24–120 h p.i.) showed decreasing Ct values from 31 at 24 h to 22 at 96 h, with a slight increase to 25.4 at 120 h (Figure 6). The highest viral titer (10^6.5 TCID₅₀/mL) corresponded with the Ct value of 23 at 72 h p.i., indicating peak replication during mid-logarithmic growth phase.

The JEV/Pig/Assam/NIVEDI-1/2025 (GI) isolate produced smaller and more variable plaques compared with the GIII reference strain JEV/eq/India/H225/2009 (H225) maintained in our laboratory. The mean plaque diameter for the reference strain was 2.68 ± 0.48 mm, whereas the GI isolate produced plaques averaging 1.88 ± 0.56 mm (Figure 7). The difference in mean plaque size between the two genotypes was statistically significant (unpaired t-test, p < 0.01), suggesting phenotypic variation between JEV genotypes I and III.

The four pig serum samples from Farm A collected on 29 May 2025 were negative for neutralizing antibodies to JEV (MNT titers <1:10) (Table 1). Of the 30 serum samples screened during June 2025 from the same village, 11 (37%) were positive for JEV IgG.