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Molecular, serological, and epidemiological characterisation of a hepatitis A virus outbreak in Maharashtra, India, 2025
For correspondence: Dr Anita Shete Aich, Hepatitis Group, ICMR-National Institute of Virology, Pune 411 021, Maharashtra, India e-mail: anitaaich2008@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Tomar S, Pise KH, Ramdasi A, Hundekar S, Deoshatwar AR, Thorat N. Molecular, serological, and epidemiological characterisation of a hepatitis a virus outbreak in Maharashtra, India, 2025. Indian J Med Res. 2026;163:671-8. doi: 10.25259/IJMR_2295_2025
Abstract
Background and objectives
Hepatitis A virus (HAV) remains a major cause of jaundice outbreaks globally, with 158.9 million infections reported in 2019 despite a 63% decline in mortality since 1990. Improved sanitation in many regions has shifted the age of primary infection from early childhood to older children and adults, increasing the risk of symptomatic and severe disease. In April–May 2025, we investigated an outbreak of HAV caused hepatitis that occurred in Rajur village, Ahilyanagar district, Maharashtra. A total of 327 suspected cases of acute hepatitis were reported, mainly affecting older children and young adults.
Methods
Suspected acute hepatitis cases were identified by clinical presentation and their epidemiological and clinical data were collected by questionnaire. Serum, stool, and potable water samples were collected and tested by anti-HAV IgM ELISA and/or real-time RT-PCR to confirm outbreak aetiology. Full HAV genome sequences were obtained from selected RT-PCR positive specimens.
Results
The age-specific attack rate for the 327 acute hepatitis cases was the highest in children aged 10-19 years (11.97%) and lowest in those ≥ 50 years. Anti-HAV IgM antibodies were detected in 32 of 45 (71.1%) available serum samples, and HAV RNA was detected in 7 of 22 (31.8%) serum and 19 of 31 (61.3%) stool samples tested. All 7 potable water samples tested negative for HAV RNA. Genomic analysis revealed strain details relevant to disease severity, providing insights into circulating HAV lineages in India.
Interpretation and conclusions
The findings highlight the need for continuous surveillance, genomic monitoring, and targeted prevention strategies to protect vulnerable, previously unexposed populations.
Keywords
Acute hepatitis
Genotype III
Hepatitis A
Mutations
Nanopore
Outbreak
Jaundice outbreaks are a major public health concern with an estimated 158.9 million infections occurring in 2019. 1 Hepatitis A virus (HAV) is an important aetiology, spreading mainly through the faecal-oral route by contaminated food and water sources. 2- 5 Early childhood infections are usually asymptomatic, but jaundice develops in ∼70% of cases in adults, with ∼1% experiencing fulminant hepatic failure. 6,7 With socio-economic development and better sanitation, a shift in age at infection from early childhood to older children and adults has occurred, resulting in increased risk of symptomatic and severe disease. 8- 10
India reflects this epidemiological transition, with recurring waterborne outbreaks being noted in Kerala, Karnataka, Punjab, and Kashmir during the last decade. 11 Kerala had 84 HAV outbreaks and 22 fatalities during 2012-2016 among hitherto unexposed older ages. 12 During 2022, Maharashtra's integrated disease surveillance programme reported 38 hepatitis A-like outbreaks, causing 19 deaths. 13
HAV, a picornavirus-like small, non-enveloped, positive-sense single-stranded RNA virus, infecting virtually 90% of children in low-and middle-income nations by 10 years of age, usually asymptotically, 14 and is environmentally sturdy. 15,16
Global burden of disease data approximates 160 million HAV infections per year, causing 26,901 deaths and 1.8 million disability-adjusted life years, with India having 19% of infections and half the global deaths. 17 Epidemiological evidence shows a transition in age-specific susceptibility from children to young adults in India and a rising incidence of symptomatic, severe, and acute hepatic failure cases. 10
An acute hepatitis outbreak occurred in Rajur village, Ahilyanagar district, Maharashtra, in April 2025 involving older children and adolescents. In the current study, we investigated the outbreak to identify its source, possible mode of transmission and to recommend appropriate control and preventive measures.
Methods
This study was undertaken by the Hepatitis Group, ICMR-National Institute of Virology, Pune, Maharashtra, India. The ethical clearance was received from the Institutional Ethics Committee for this study.
Background
Rajur village has a population of 10,046 with 5,238 males and 4,808 females, as per the Census India report 2011, with an estimated population of 14,400 in 2025. As per the health officials, there had been no outbreak of acute hepatitis in the last 10 years in Rajur.
Sample collection
Following an apparent rise in acute hepatitis cases observed by the medical officer at primary health centre (PHC) Vitha, Rajur village, a rapid response team (RRT) was constituted to conduct field verification on April 23 and April 24, 2025. Cases were defined as follows:
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Suspected case: Resident of Rajur with acute onset of fever or malaise plus jaundice or dark urine since 11 April 2025.
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Confirmed case: Suspect with serum anti-HAV IgM-positive by ELISA.
Inclusion criteria: Cases fulfilling the case definition of a suspected acute hepatitis case, and willing to participate in the study
The Hepatitis Group at ICMR-National Institute of Virology (NIV), Pune were informed regarding this apparent increase in acute hepatitis cases on May 1, 2025, when human and environmental samples (10 serum samples, 10 stool samples, 3 water samples) were sent for further testing and confirmation of aetiology. The ICMR-NIV team went on the field to Rajur Village for further investigation and sample collection on May 3, 2025.
Human specimens
A multidisciplinary team comprising of an epidemiologist and a medical microbiologist, technologists from ICMR-NIV actively participated in field investigation of the outbreak. A structured questionnaire was administered to the cases where information on clinical symptoms, sanitation, toilet facilities, clustering, food source, and drinking water source was documented. Sample collection, patient interviews and recording of clinical details was done from patients admitted at the Rural hospital Rajur and from the suspected cases visiting the make-shift sample collection site at a Marathi school. A total of 35 serum samples were collected on field i.e. from 12 admitted patients (with 10 paired stool samples) and 23 serum samples from suspected cases. All human specimens collected on field were stored at the Rural hospital at Rajur village at 4°C in a refrigerator and were transported to the ICMR laboratory in thermocol boxes lined with ice packs.
Potable water sample
Households in the affected village primarily received drinking water from Nilawande Dam, directly supplied to them without any form of pre-treatment. Additionally, the water filtration plant had been non-operational for over a year. A three-day religious ceremony (jatra) took place near the Dam from April 11-13, 2025, where there were only four toilets each for males and females, but footfall was 10,000-15,000, along with open defecation. Approximately 10 days after the event, a noticeable increase in acute hepatitis cases was reported.
Water samples (n=4) were collected during the field visit i.e. (i) main water storage tank of village; (ii) jackwell receiving water from the Nilawande dam; (iii) water filtration plant (before filtration); and (iv) water storage tank situated at patients home. Five litres of water was collected from each suspected site, on field.
Water sample concentration
All water samples were concentrated using the sea salt and milk powder flocculation method, as described previously. 18
Serology
Serum samples were tested for the presence of anti-HAV IgM and anti-HEV IgM antibodies as per manufacturer's instructions (Wantai, Taiwan). Additionally, serum samples were tested for hepatitis B surface antigen (HBsAg) (Monolisa TM HBsAg ULTRA Bio-Rad, France) and anti-HCV antibodies using ELISA kits from J. Mitra and Co. Pvt. Ltd. (New Delhi). 19
Real time RT PCR
RNA was extracted from both serum and stool samples. Stool samples were processed to prepare 10% suspensions in 1X PBS. 140 uL of stool/serum was used for RNA extraction (e.g., QIAamp Viral RNA Mini Kit, Qiagen, Germany), following the manufacturer’s instructions. Extracted RNA was eluted and stored at −80°C until further analysis.
Quantitative real-time RT-PCR (RT-PCR) was performed to detect HAV RNA copy number using an in-house assay targeting the 5’NCR region of the HAV genome, as described previously 20 and the limit of detection (LOD) of the assay was 100 RNA copies/mL.
Library preparation and sequencing
HAV-positive RNA was reverse-transcribed into cDNA with random hexamers and high-fidelity reverse transcriptase, with full-length genomes amplified through overlapping primers (∼500 bp) based on aligned GenBank references using PrimalScheme software. Amplicons were purified, pooled, and ready for sequencing to obtain complete and precise genome coverage. 21 HAV-positive amplicons were cleaned with AMPure XP beads (Beckman Coulter) and measured using a Qubit 4 Fluorometer. Sequencing libraries were prepared with Oxford Nanopore ligation kit (SQK-LSK109) and barcoded (SQK-NBD114.24) for multiplexed sequencing. Libraries were run on a MinION Mk1C (R10.4.1), and consensus genomes prepared with Commander (Genotypic India Pvt. Ltd.) were mapped to reference HAV strain FJ360733 (1995). 22
Data analysis
Nanopore sequencing (∼17 h) produced ∼4.72 million reads (3.43 Gb) with an N50 of 645 bp and >99% genome coverage in all samples. Reads were trimmed and demultiplexed using Porechop and filtered with NanoFilt (Q ≥ 7). Quality reads were mapped to reference HAV genomes through Minimap2. Consensus sequences were improved with Medaka and Nanopolish. Multiple sequence alignment was conducted with MAFFT, and phylogenetic analysis was carried out in MEGA 6 using neighbor-joining and 1,000 bootstrap replicates. Genotypes were designated from full-genome or VP1/2A sequences against reference strains, offering molecular epidemiologic information on outbreak origin and transmission patterns. 23
Results
From 22nd April 2025 till 6th May 2025, a total of 327 suspected jaundice cases were identified, with preponderance in the age group of 10 to 19 years. Females and males were equally represented and suspected cases exhibited various combinations of classic symptoms suggestive of hepatitis A infection, including fever, anorexia, and abdominal pain, yellowish discoloration of sclera and skin, and dark-coloured urine.
The index cluster appeared on 22 April, 11 days after the religious fair, with two distinct peaks (24 and 28 April) suggestive of a common-source exposure followed by limited person-to-person spread. A total of 76 human clinical specimens—including 45 serum samples and 31 stool samples—along with seven water samples were received and tested at the Hepatitis group at ICMR-NIV Pune (Supplementary Table). Of these, 35 serum and 22 stool samples were collected on field by the ICMR team on May 3 and 4, 2025, and the rest were sent by the health officials on May 1, 2025, for confirmation of aetiology.
Analysis of the structured questionnaires revealed that the cases were predominantly clustered in specific households and neighbourhoods within Rajur village. No single common food source was identified; however, all affected households reported consuming water from the same distribution system, directly from tap in the households without any form of pretreatment. The filtration plant that receives the Nilawande dam water, was only operational from April 28, 2025.
Descriptive findings
Age-specific attack rate among the 327 suspected acute hepatitis cases was highest in older children aged 10-19 years (11.97%), and lowest among adults aged ≥40 years (≤0.45%) ( Fig. 1, Table I). Among the 312 cases whose disease severity information was available, 275 were mild (88%), 35 moderate (11%) and two were severe cases who succumbed to the infection. Both mortalities developed acute liver failure within 72 h of admission; neither had any underlying liver disease. Overall case fatality rate (CFR) reported during the outbreak was 0.61%. Epidemic curve of the outbreak is shown in Figure 2.
| Age group (yr) | No. of cases | Population of village (2024) | Attack rate (%) |
| <10 | 10 | 1745 | 0.57 |
| 10 to 19 | 242 | 2022 | 11.9 |
| 20 to 29 | 38 | 2459 | 1.55 |
| 30 to 39 | 19 | 1767 | 1.05 |
| 40 to 49 | 7 | 1579 | 0.44 |
| 50 to 59 | 5 | 1158 | 0.40 |
| 60+ | 7 | 1556 | 0.45 |
| Total | 327 |
Serological findings
Among the 45 serum samples tested, 32 (71.1%) were positive for anti-HAV IgM antibodies using enzyme-linked immunosorbent assay (ELISA) indicating recent infection with HAV. Presence of anti-HAV IgM antibodies is a known sign of an acute or recent infection, usually appearing early in the illness and can be detected for several weeks to a few months, after exposure. Detection of anti-HAV IgM in more than two-thirds of the samples suggested a recent or ongoing outbreak of hepatitis A in the population studied. All serum samples were negative for antibodies for anti-HEV IgM antibodies, anti-HCV antibodies and HBsAg.
Molecular detection and genotyping
Real-time RT-PCR was used to further analyse 22 serum samples with sufficient quantity for testing, while the remaining samples could not be processed due to limited quantity available. Seven of these 22 samples tested positive for HAV RNA, indicating active viremia and continued viral replication.
HAV RNA was detected in 19 (61.3%) stool samples with RNA copy numbers ranging from 102 to 107 calculated genome copies. Based on partial genome sequencing of representative RNA-positive samples, aetiological agent of the current outbreak was confirmed as Hepatitis A virus genotype IIIA. Nanopore sequencing was carried out with > 99% genome coverage for four samples and the sequences were submitted to GenBank with accession numbers PX136630–PX136633. Amino acid substitutions observed in structural proteins VP2 and VP3 of HAV-positive samples from Rajur village are shown in Table II.
| Gene name | AA position | AJ299464 (Prototype Norway) | FJ360735 (Indian isolate) | 2516775 | 2516784 | 2517003 | 2517000 |
| VP2 | 119 | F | F | V | V | F | F |
| VP3 | 97 | C | W | C | C | C | C |
| 190 | S | S | A | A | A | A | |
| 2A | 21 | E | E | D | D | D | D |
| 37 | E | E | D | D | D | D | |
| 52 | E | G | E | E | E | E | |
| 2B | 201 | E | E | D | D | D | D |
| 230 | S | S | STOP CODON | S | S | S | |
| 245 | M | M | M | M | L | M | |
| 2C | 132 | P | T | T | T | T | T |
| 3A | 29 | S | T | S | S | S | S |
| 70 | S | S | T | T | T | T | |
| 3B | 4 | H | H | H | Q | Q | Q |
| 3C | 87 | L | V | L | L | L | L |
| 3D | 426 | N | N | S | S | S | S |
Phylogenetic analysis of HAV genotype IIIA sequences revealed two distinct clusters ( Fig. 3 ). One cluster included strains from India that showed close genetic relationships with sequences from Norway (AJ299646), Japan (AB973400), and Korea (JQ655151). Percent nucleotide identity between Rajur village outbreak sequences and reference strains was remarkably high: 99.96% with Norway and Korea isolates and 99.95% with the Japan isolate, indicating strong genetic conservation and ongoing circulation of related strains across different geographic regions.
Environmental samples
Water samples (n=7) were taken from different areas of the impacted area, to identify the potential source. Real-time RT-PCR was used to test for the detection of HAV RNA. All seven water samples tested negative for HAV RNA, indicating that there was no detectable viral contamination in those particular sources at the time of sampling.
Discussion
Using serological and molecular methods, our investigation identified HAV as the causative agent of the Rajur village outbreak. The acute viral hepatitis clinical pattern, which involved children and adolescents mostly, was indicative of continued transmission of the virus in an environment moving from high to intermediate endemicity. Genotyping identified the strain to be of genotype IIIA, which is known to circulate extensively throughout India. The presence of sequence heterogeneity in Rajur village isolates, as well as certain non-structural mutations, implies localised viral evolution and potential multiple introductions instead of an outbreak of single-source origin. Contaminated drinking water or water used for food preparation and poor sanitation are known risk factors for HAV transmission from the faecal-oral mode, particularly in places with poor infrastructure and water quality.
Although the usual incubation period for Hepatitis A infection ranges from 15 to 50 days (mean 30 days), a shorter incubation period of as few as 10 days has been documented in the literature. 5 Considering this, the probability of the index cluster appearing 11 days after the religious fair or jatra, make it a plausible cause of the current outbreak, where open defecation was practiced due to limited toilet facilities, and dam water was directly supplied to the households without any form of pretreatment. Also, it has been reported that high doses of virus or inoculum can lead to a shorter incubation period in Hepatitis A infection. 24 All water samples were negative for HAV RNA, indicating there was no detectable viral contamination at the time of sampling, but possibility of waterborne transmission remains high, considering clustering of cases in a single village. Negative water sample results could be attributed to initiation of the water treatment process shortly after appearance of the index cluster, but it was noted during field investigation that the filtration plant at the dam was non-operational since the past 1.5 years.
Indian and Southeast Asian studies have genotype IIIA as the predominant strain circulating among sporadic cases as well as outbreaks in communities. 25 The amino acid substitutions observed in structural proteins were not located within conformational epitopes known to be involved in receptor binding or virus neutralisation, 26 suggesting that antigenic properties of the virus, particularly those relevant to immune recognition and vaccine response, likely remain unchanged. Structural and non-structural proteins both play distinct roles in viral biology, with structural proteins involved in virion assembly and entry, while non-structural proteins are involved in replication and enzymatic functions. Interestingly, 2B, 2C, and 3A mutations increase replication efficiency, while 3C protease and 3D RNA polymerase mutations affect genome replication and accuracy in vitro. These changes can affect viral fitness and transmissibility, emphasising the importance of specific functional studies. Specifically, mutations in 2B, 2C, and 3A regions are known to enhance viral replication efficiency in cultured cells, while changes in 3C protease and 3D RNA-dependent RNA polymerase contribute to genome replication and overall replication fidelity in vitro. 27 These molecular features may influence viral fitness and transmissibility, underscoring the need for further functional studies.
Non-structural mutations were also reported in Indian HAV isolates, reflecting adaptive evolution under local pressures. Nonetheless, the presence of a premature stop codon in the 2B region of an individual Rajur village isolate is rare and can be indicative of a defective or attenuated genome occurring together with replication-competent strains—a finding not often encountered in earlier Indian investigations. It is interesting to note that the patient infected with this suspected defective or attenuated HAV strain had a mild illness and recovered completely under home care. Previous studies have highlighted 2C, 3C, and 3D mutations for their contribution to replication competence and fidelity of the polymerase; our results extend these findings by locating specific amino acid substitutions that could impact viral fitness and transmission patterns. 28
HAV being an atypical Picornaviridae grows poorly in cell culture and produces no cytopathic effects, thus neutralisation assays are not recommended for its diagnosis, and is usually confirmed by serological and molecular techniques. Titrations are laborious, time-consuming, and are commonly done on monkey kidney or human fibroblast cell lines using attenuated strains of HAV highly adapted to grow in these cell cultures, containing a significant number of adapting mutations. 29 Implementation of next-generation sequencing allowed for identification of distinctive mutations, which informed local viral evolution. Limitations of our study include few sequenced isolates and inability to pinpoint the specific environmental source because of delayed water sampling, post-control interventions, although the religious fair held in the village along the water dam is suspected to lead to the current outbreak. More comprehensive genomic sampling, along with contemporaneous environmental sampling, would more precisely inform transmission pathways and evolutionary patterns.
This outbreak highlights that integration of serological, molecular and genomic tools help not only to identify the aetiology with accuracy, but also to uncover local viral evolution, relevant to the transmission dynamics. Detection of region-specific mutations can guide assay development, consolidate laboratory diagnosis, and optimise external quality assessment programmes. For clinicians, it reinforces the importance of early recognition of hepatitis A, especially in older children and adolescents, an age group increasingly impacted during endemic transition, highlighting timely case detection, providing supportive care, and rapid public health action remaining central to prevent complications and limiting community-wide spread. At the policy level, it emphasises the necessity of enhancing water and sanitation infrastructure, implementing regular monitoring of drinking water quality, and exploring the implementation of targeted hepatitis A vaccination in epidemiologically transitioning areas. 26
Author contributions
ST: Conceptualisation, methodology, formal analysis, investigation, field work, manuscript writing; KP: Conceptualisation, methodology, formal analysis, investigation, field work, manuscript writing; AR: Laboratory work; SH: Laboratory work; ARD: Conceptualisation, methodology, manuscript writing; NT: Laboratory work; PJ: Field work, laboratory work; PA: Laboratory work; PK: Laboratory work; BBN: Investigation, field work, manuscript writing; BVT: Supervision, conceptualisation, methodology, data curation, manuscript writing; ASA: Supervision, conceptualisation, methodology, data curation, manuscript writing. All authors have read and approve the final printed version of the manuscript.
Acknowledgment
Mr. K.D. Ramaih for sample processing work and data entry and Mr. Machindra Karanjwane for helping in sample collection during field work.
Financial support and sponsorship
The study received institutional funds from ICMR-National Institute of Virology, Pune, Maharashtra, India.
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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