Molecular Evolution of Hepatitis B Virus in India: Insights into Genotype Diversity and Recombination
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Background Hepatitis B virus (HBV) is a major public health problem in India, with considerable genetic variability arising out of evolutionary selection pressure, recombination events and reverse-transcription stage in its life cycle. This affects the transmission patterns, disease chronicity, and treatment response of the virus. Although HBV genotypes A, C, and D have been described in India, detailed whole-genome sequence data are scarce. Methods Residual HBsAg positive samples (n = 1963), received for inter-laboratory quality control or for referral diagnostic testing between 2006 and 2023 were screened, and n = 172 of these were positive for HBV DNA by realtime PCR. HBV DNA positive samples with high viral load (n = 32), collected from nine states of India, were used for whole-genome sequencing. DNA extracted these serum samples was subjected to whole-genome sequencing (WGS) on the Oxford Nanopore Technology (ONT) MinION platform. Genotype-specific tiled amplicons (~ 1000 bp) were used for library preparation. Consensus genomes were assembled, genotyped, and phylogenetically analysed using MEGA v11.0. Recombinant sequences were characterized further by SimPlot v3.5.1 boot scanning and confirmed with χ² testing of phylogenetically informative sites. Results HBV genotype D (70.96%) was found to be common genotype in several Indian states, with genotype A (3.22%) present in central and southern states and genotype C (6.45%) in the northeast. Notably, five samples showed evidence of recombination events, including genotypes A, C, and D. There were two recombinants in West Bengal, and one each in Chhattisgarh, Assam, and Maharashtra. SimPlot analysis of representative isolates showed clearly demarcated recombination breakpoints. Phylogenetic reconstruction also attested to the mosaic character of these genomes. Conclusions The present study emphasizes the genetic heterogeneity and evolutionary dynamics of HBV in India, and recombination as a key mechanism adding to viral diversity. The success in utilizing ONT for WGS of HBV directly from serum samples illustrates its utility for scalable genomic surveillance. The initiation of a nationwide HBV sequencing program will be pivotal in tracking emerging variants, informing therapeutic interventions, and facilitating India's role in the global HBV elimination drive.
