According to the latest WHO Hepatitis B fact sheet, an estimated 254 million people worldwide were living with chronic hepatitis B virus (HBV) infection in 2022, highlighting the significant worldwide impact of this disease. India alone harbours a significant portion of this burden, with approximately 40 million people living with chronic HBV infection (World Health Organization, 2025). In India, HBV vaccination was introduced into the Universal Immunisation Programme in 2011, and national coverage with the three-dose HBV vaccine has steadily increased, exceeding 85–90% in recent years, although regional and sub-national disparities persist, particularly in northern India (World Health Organization, 2019). Concurrently, India has a substantial HIV burden, with approximately 23.49 lakh individuals living with HIV/AIDS (HIV Facts & Figures, 2019). In northern India, HBV prevalence is particularly high, and HBV-HIV co-infection rates have been reported to reach 7.1% (HIV). Understanding the genetic diversity of HBV in both HBV mono-infected and HIV-HBV co-infected individuals is essential for enhancing clinical management, improving treatment outcomes, and informing public health strategies.

Classically, there are four primary serotypes based on the major antigenic determinants of the surface antigen (HBsAg): adw, adr, ayw, and ayr. However, with more detailed serological studies, up to nine serological variants have been described (e.g., adw2, adw4, adrq+, adrq–, ayw1, ayw2, ayw3, ayw4, and ayr) based on finer differences in HBsAg antigenicity (Datta, 2008; Mlewa et al., 2025). Because of its distinctive life cycle that involves an error-prone reverse transcriptase for replication, the virus continually evolves, leading to significant genetic diversity in genotypes, sub-genotypes, and mutations (Zhang et al., 2016). HBV genomes have significant sequence variation, comprising at least 10 genotypes (A to J) and over 40 known genetic subtypes (Kramvis et al., 2005). In India, genotype D is the most widespread, followed by genotype A, with genotype C being the least common in the north region (Sami et al., 2013; Chen et al., 2023).

In immunocompetent individuals, most acute HBV infections resolve spontaneously; however, progression to chronic infection is strongly age-dependent, occurring in approximately 90% of infants infected within their first year, ~30% of children infected between 1 and 5 years of age, and only 5–10% of individuals infected after 5 years of age (Pattyn et al., 2021). In contrast, people living with HIV (PLWH) have a lower clearance rate, leading to persistent HBV replication and worse outcomes due to immunosuppression. Also, there is increasing evidence that certain mutations in the HBV viral genome are higher in HIV-HBV co-infected individuals and favour the rapid progression of liver disease in these individuals (Singh et al., 2017).

Despite the high prevalence of HBV infection in North India, extensive data on regional HBV molecular diversity, specifically regarding surface gene variants, serotypes, and genotype distribution in HBV co-infected with HIV, is scarce. Due to the significant HIV prevalence in this area and the influence of HIV-related immunosuppression on HBV evolution, region-specific molecular characterisation is crucial for enhancing diagnostic accuracy, directing antiviral treatment, and guiding targeted public health measures. This study aims to characterize the genetic variability of the HBV surface gene, genotypes, sub genotypes, serotypes among HBV mono-infected and HBV-HIV co-infected patients in North India.

This was a hospital-based cross-sectional observational study conducted at a tertiary care centre in North India.

Among the 100 HBV-HIV co-infected patients the mean age was 36.59 years (Female: Male : Transgender = 29:70:1), while HBV mono-infected patients had a mean age of 34.92 years (F:M = 19:31) as shown in Table 1. Conventional PCR successfully amplified the HBV surface (S) gene region (nt 107–718) in 19% of co-infected and 40% of HBV mono-infected samples (Figure 2), which were further confirmed by real-time PCR and subjected to Sanger sequencing. Patients in whom HBV surface gene amplification was successful had significantly higher HBV DNA levels and higher HBeAg positivity compared with those in whom amplification failed, both among HIV–HBV co-infected and HBV mono-infected individuals as shown in Supplementary Table 2. In contrast, age, liver enzyme levels (ALT, AST, alkaline phosphatase), APRI, FIB-4 scores, and ART duration did not differ significantly between sequenced and non-sequenced patients.

In this study, the HBV–HIV co-infection rate among ART-enrolled patients was 7.2%, which is comparable to reports from hospital-based studies in North India involving ART clinic populations, where prevalence rates of 5.3% (Gupta et al.), 9.9% (Jain et al.), and 11.3% (Saha et al.) have been documented, while a higher prevalence of 22.2% was reported by Sud et al., likely reflecting differences in study population characteristics, sampling strategies, and clinical settings (Chauhan et al., 2006; Gupta and Singh, 2006; Jindal et al., 2008; Jain et al., 2009; Saha et al., 2013; Chakravarty and Pal, 2015) (Gupta and Singh, 2006; Ruiz-Tachiquín et al., 2007; Jindal et al., 2008; Jain et al., 2009; Tamura et al., 2011; Chakravarty and Pal, 2015). Although this prevalence is lower than that reported from some high-endemic regions globally, it remains clinically and programmatically significant, given the well-established association of HBV–HIV co-infection with accelerated liver disease progression, increased risk of hepatocellular carcinoma, and greater complexity of antiviral management. In the era of widespread HAART, chronic viral hepatitis has emerged as a leading non-AIDS-defining cause of long-term morbidity and mortality, underscoring the importance of routine HBV screening and integrated management strategies within ART programs.

Consistent with previous studies, the mean CD4⁺ T-cell count was lower in HIV–HBV co-infected patients than in HIV mono-infected individuals. Reduced CD4⁺ T-cell counts reflect impaired immune surveillance, which may permit enhanced HBV replication and persistence, thereby contributing to adverse liver-related outcomes in HIV-HBV co-infected individuals.

The prevalence of HBeAg among HIV-HBV co-infected patients in this study was 17% as compared to HBV mono-infected patients in which it was 10%, however, this difference was not statistically significant. Koli et al. (Koli et al., 2016) reported a prevalence of 5.8% in HBV mono-infected individuals, whereas a study conducted in South India (Chandra et al., 2013) revealed a positivity rate of 21.4% among HIV-HBV co-infected patients. Given that HBeAg is a marker of active viral replication, the overall low HBeAg positivity in this cohort suggests that a substantial proportion of patients may have undergone HBeAg seroconversion, possibly influenced by immune status and antiretroviral therapy. Among the 39 HBV sequences analysed, genotype D predominated (89.7%), while genotype A accounted for 10.3%, a distribution broadly consistent with reports from North India (Thakur et al., 2002; Sami et al., 2013; Pacholi et al., 2022). Genotype A is common in northwestern Europe, while genotypes B and C dominate east Asia. However, genotype D is widespread (Thakur et al., 2002). Genotype D dominates northern, western, and southern India and is also found in Saudi Arabia, Iran, Afghanistan, Pakistan, and Turkey. The prevalence of genotype D is clinically significant, as this genotype correlates with reduced rates of spontaneous HBeAg seroconversion, extended immune-active phases, and an increased probability of progressive liver disease in comparison to genotype A. Genotype D also has more nucleotide variation in the surface gene, which could affect how HBsAg is expressed, how well it can escape the immune system, and how sensitive it is to laboratory tests. Conversely, genotype A, prevalent in northwestern Europe and certain regions of Africa, is associated with elevated HBeAg positivity rates, enhanced responses to interferon-based therapy, and expedited seroconversion, possibly indicating variations in viral replication dynamics and host–virus interactions. The significant prevalence of genotype D in this study has critical implications for disease progression, treatment response, and long-term clinical outcomes within this population. From an evolutionary and epidemiological perspective, the prevalence of genotype D in northern India likely signifies historical transmission dynamics and population migrations throughout the Middle East and South Asia, highlighting the necessity of region-specific molecular surveillance. Within genotype D, subgenotypes D1 and D3 were identified in this study. Subgenotype D1 is largely reported from the Mediterranean and Middle Eastern regions, whereas other genotype D subgenotypes (D2–D8) exhibit larger distribution across Africa, Eastern Europe, Australia, Indonesia, and India, reflecting a strong geographical pattern of HBV evolution. The finding of D1 and D3 in North India is consistent with regional molecular epidemiology and support historical and ongoing population movement impacting viral dissemination. All genotype A isolates belonged to subgenotype A1, which is the dominant A subgenotype reported from South Asia and parts of Africa (Ohba et al., 1995).

At the serological level, serotype ayw2 predominated, being present in nearly all genotype D strains, while serotype adw2 was exclusively associated with genotype A isolates. This distribution is consistent with well-established genotype–serotype linkages, whereby genotypes A, C, D, and F are typically associated with serotypes adw2, adr, ayw2/3, and adw4, respectively (Datta, 2008). In India, ayw and adw serotypes are most prevalent, with adr reported less frequently, particularly from southern regions (Thakur et al., 2002). The predominance of genotype D with serotype ayw2 and genotype A with serotype adw2 in the present study therefore aligns well with known regional patterns in North India.

HBV serotypes are determined by specific amino acid residues within the HBsAg, notably at positions 122, 127, 134/140, 159, 160, 177, and 178, which explains the close linkage between genotype and serotype. The consistent association of genotype D with serotype ayw2 and genotype A with serotype adw2 observed in this study reinforces the stability of this genotype–serotype relationship and supports the validity of the molecular and serological classification employed (Ohba et al., 1995).From an evolutionary perspective, the detection of genotype A (A1/adw2) in this population also mirrors patterns reported in Europe. Historical migration and long-standing population exchange between India and the United Kingdom over the past several centuries may have contributed to the introduction and persistence of genotype A strains in the Indian subcontinent. Together, these findings underscore the necessity of integrating genotype, subgenotype, and serotype data to better understand HBV transmission patterns, viral evolution, and region-specific clinical implications (Kao, 2002; Kramvis, 2014).

Mutations within the HBV surface (S) gene, particularly in the major hydrophilic region (MHR) and its ‘a’ determinant (amino acids 124–147), are of major clinical and public health significance. This region constitutes the primary target of neutralizing antibodies induced by both natural infection and hepatitis B vaccination. Amino acid substitutions within the ‘a’ determinant—most notably G145R, but also mutations at positions D144, P120, T126, and Q129—can alter the conformational epitope structure of HBsAg, resulting in reduced antibody binding, vaccine escape, and false-negative results in HBsAg-based diagnostic assays. The clinical implications of these mutations are substantial, as they may lead to occult HBV infection, ongoing viral transmission despite vaccination, and underestimation of true HBV prevalence due to diagnostic escape. From a public health perspective, the circulation of S gene escape variants underscores the need for continued molecular surveillance, periodic evaluation of diagnostic assays, and potential updates to vaccine design to ensure sustained effectiveness of HBV control strategies, particularly in high-burden settings (Carman et al., 1990; Banerjee et al., 2006; Salpini et al., 2015).In this study, nucleotide substitutions within the “a” determinant were observed in 7 (36.8%) HBV-HIV co-infected patients. No such mutations were identified in HBV mono-infected individuals. Of these, two were nonsynonymous (amino acid-altering), while five were synonymous. In addition to impairing antibody-mediated neutralization, these mutations pose challenges for HBV control by potentially undermining the effectiveness of passive immunisation strategies and reducing the sensitivity of routine HBsAg-based diagnostic assays. Their emergence in HIV-HBV co-infected populations underscores the importance of sustained molecular surveillance in this group to accurately monitor viral evolution and ensure the continued effectiveness of preventive and diagnostic interventions (Pondé RA de and Amorim G de, 2024). Additional mutations in pre-S and S regions were linked to higher ALT/AST levels and elevated APRI and FIB-4 scores, suggesting a potential association with liver disease progression. Specific mutations—preS1-A482C, S-A644C, and S-G684A—were observed in patients with high fibrosis scores, though no statistically significant association could be confirmed due to limited sample size. Other mutations such as S-C198A, S-G195T, preS-C162T, and preS-A163G were significantly associated with deranged liver function tests and were more common in genotype A cases, warranting further investigation in larger cohorts. These mutations may impair antibody recognition, leading to vaccine escape, failure of passive immunisation, and false-negative serological results and suggest the need for continued molecular surveillance in HIV-HBV co-infected populations. Interestingly, pre-S amino acid insertions identified in this study did not correlate with liver disease severity, unlike previous reports (Chen, 2018). HBeAg positivity was associated with the S-G189A mutant, suggesting a possible role in viral replication or immune modulation.

While several of the detected S and pre-S mutations have established roles in immune escape and liver disease in other settings, the associations observed in this study should be interpreted cautiously. They highlight potential mutation hotspots in HIV–HBV co-infection that warrant further investigation using larger, longitudinal cohorts and functional assays.

The limitations of this study include the small sample size, small S-gene coverage, lack of longitudinal follow-up, and the use of a genotyping assay designed to detect HBV genotypes A–H, which may not identify the rare genotypes I and J. However, given the absence of reports of these genotypes from northern India, their omission is unlikely to materially affect the interpretation of the findings. The genotype distribution, serotype frequencies, and prevalence of surface gene mutations reported here may not be fully representative of the entire study population, particularly in patients with low-level viremia. However, the observed predominance of genotype D and the higher frequency of ‘a’ determinant mutations in HBV–HIV co-infected patients remain clinically relevant, as individuals with higher viral loads are at increased risk of transmission, disease progression, and diagnostic or vaccine escape. Furthermore, the use of consecutive, non-probability sampling may limit the generalizability of the findings beyond the hospital setting, as the study population may not fully represent the broader community of HBV-infected individuals.

Shared pathways of transmission and synergistic effects of both viruses have an impact on morbidity and mortality of HIV. Screening of HBV should be included in the investigations to be done pre-HAART. This study highlights genotype D predominance and a higher frequency of immune escape mutations in HBV-HIV co-infected patients. The exclusive presence of serotype ayw2 across all genotype D isolates and the detection of S gene mutations suggest the need for continued molecular surveillance in HIV-HBV co-infected populations.