Whole-Genome Sequencing of Hepatitis B Virus Genotypes E and A in Zambia Reveals Limited Viral Diversity in HIV Coinfection

Abstract

Background

The molecular characteristics of hepatitis B virus (HBV) in Africa, including the impact of HIV coinfection, are poorly understood.

Methods

We performed whole-genome sequencing (WGS) on biospecimens collected before antiviral therapy in a well-characterized cohort of adults with HBV in Zambia, enriched for HIV coinfection (HBV/HIV). We assessed the frequency of basal core promoter (BCP) and precore variants, substitution frequencies, and the ratio of nonsynonymous to synonymous substitutions (dN/dS ratios), a surrogate for selection pressure.

Results

Among 215 participants (median age, 33 years; 36% e antigen [HBeAg] positive, 35% with HBV/HIV), 114 (53.0%) had viral genotype E (gtE), and 101 (47.0%) had genotype A (gtA), subgenotype 1. BCP and precore variants, associated with HBeAg negativity, were more common with increased age, in the absence of HIV, and with gtE. Distinct from gtA, gtE had dN/dS ratios that were increased in the core vs polymerase region. Low dN/dS ratios were observed in HBV/HIV, especially at the lowest CD4 T-cell frequencies. Sequences from acute HBV infection as well as from 5 participants with chronic HBV/HIV who cleared hepatitis B surface antigen early during tenofovir-based antiretroviral therapy showed remarkably low dN/dS ratios.

Conclusions

HBV gtE exhibited distinct substitution patterns compared with gtA, and HBV/HIV was associated with reduced HBV sequence diversity, consistent with impaired immune pressure.

A better understanding of the hepatitis B virology in Africa is needed to achieve viral elimination. In this paper, we report the analysis of 215 whole genome sequences from adults with treatment-naïve acute and chronic HBV and HBV/HIV coinfection in Zambia. Results shed new light on genotypic differences (E versus A1), as well sequence features that may be associated with surface antigen seroclearance.

Graphical Abstract

Graphical Abstract.

The Africa region should be a major focus of global hepatitis elimination because of its high prevalence and incidence hepatitis B virus (HBV) infection and significant gaps in deploying evidence-based strategies to control viral transmission, mortality, and morbidity. To support viral elimination, a better understanding of HBV virology is needed in Africa and in general [1]. HBV in Africa may have important viral characteristics that are distinct from other regions. For example, HBeAg seroconversion from positive to negative, signaling a major transition in virus–host interactions, occurs at a significantly younger age in Africa compared with Asia [2–4]. HBV-related HCC has been reported to occur at a younger age in Africa [5], possibly underpinned by variants of genotype A (gtA) [6, 7]. Unfortunately, the major HBV genotype in Africa, genotype E (gtE), is particularly neglected, with conflicting reports on whether its viral characteristics drive high mother-to-child transmission in the region [8]. While rarely seen in non-African sequencing studies [9], HBV gtE has been characterized by lower sequence diversity compared with other HBV genotypes, possibly as a consequence of its more recent emergence in West Africa ∼150–200 years ago [10]. Previous analyses of gtE often used partial viral sequencing and rarely included comparable populations (eg, shared ethnic background and environment) with another genotype.

HBV in Africa is also characterized by a high burden of coinfections, including ∼2 million individuals with HIV-1 (HBV/HIV), which is associated with a higher rate of HBV chronicity and faster liver disease progression [11, 12], yet comparisons of HBV sequence features in HBV/HIV and HBV alone are scarce. Compared with HBV alone, HBV/HIV has been reported to have different HBV genotype distribution and mutational frequencies, including those associated with altered HBeAg production, and less HBsAg clearance [13–15]. Surprisingly, during the initial phase of treatment of HBV/HIV with tenofovir-based antiretroviral therapy (ART), HBsAg seroclearance appears to be higher than expected [16], a phenomenon that is poorly understood. High rates of HBsAg loss make HBV/HIV a potentially instructive clinical stage for understanding the host and viral mechanisms of HBV cure.

In this paper, we use whole-genome sequencing (WGS) to analyze HBV in a large and well-characterized cohort of adults in Zambia, where both HBV gtE and gtA circulate [17]. Zambia has a 6% adult prevalence of chronic HBV [18]. Compared with sequencing a limited number of genes, WGS provides higher-resolution assessment of viral evolution across different genomic regions. This facilitates understanding of the genomic elements of selection pressure whether from nucleos(t)ide analogs (NAs), other therapies, natural immunity, or vaccines. The Zambian cohort uniquely includes people with chronic HBV alone, HBV/HIV, and acute resolving HBV infection. Furthermore, the cohort routinely ascertains HBsAg seroclearance among people with HBV/HIV taking tenofovir-based therapy, at a rate of 9% at 2 years and 15% at 5 years of treatment [19]. In this paper, we describe viral genotypes, molecular epidemiology, drug resistance, and viral diversity in the context of HBV/HIV, in acute HBV, and among people with chronic HBV/HIV that evolved to HBsAg loss during treatment. We hypothesize that gtE has distinct viral genetics from gtA and that viruses captured during acute and HIV coinfection, particularly in those who subsequently clear sAg, have lower viral evolution due to less immune selection pressure.

METHODS

HBV Cohort in Zambia

We applied WGS to plasma and serum samples from a longstanding HBV cohort in Zambia. At inception in 2013, cohort eligibility included newly diagnosed adults (age 18+ years) with HBV/HIV, defined as HIV-1/2 antibody and HBsAg positive in blood, without ART or who had received up to 1 month of antiviral therapy, and were not known to have hepatitis C. From 2016, eligibility expanded to include HBV alone (HBsAg-positive, HIV-negative) irrespective of treatment history. At enrollment, in addition to demographic characteristics and district of birth, we assessed HBV DNA (Roche cobas or Cepheid Xpert), liver enzymes (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]), HBeAg (Diapro or Diasorin), hepatitis delta antibodies (Diapro or Diasorin), and we stored aliquots. In those with HBV/HIV, we also determined CD4 T-cell count and plasma HIV RNA concentration. Acute HBV was defined by hepatitis B core immunoglobulin M positivity in a participant with a typical clinical presentation. For this analysis, we excluded individuals with treatment of HBV alone before enrollment, unknown or undetectable (<10 IU/ml) HBV DNA at enrollment, insufficient stored samples, and/or enrollment HBV DNA levels of 10-500 IU/mL.

Patient Consent

The study was approved by the ethics committees of University of Zambia and University of Alabama at Birmingham. All participants provided written informed consent.

Amplification and Sequencing of HBV and HIV

Viral nucleic acid from 300μL of serum or plasma was extracted automatically using the Maxwell RSC Blood DNA Kit on a Maxwell RSC 48 Instrument (both Promega), and the complete HBV genome was amplified in 2 fragments as previously described [20]. In brief, 2-step nested polymerase chain reactions were performed for the core region (nt 1683-nt 2399; 717 bp according to the reference genome NC_003977.2) and the polymerase region (nt 2299-nt 1798; 2682 bp according to the reference genome NC_003977.2). Amplicons were pooled, and a library prep for Oxford Nanopore sequencing was done. Libraries were sequenced on R.10.4.1 flowcells using the R10.4.1 chemistry. Basecalled HBV reads were filtered by the ARTIC pipeline (version 1.2.4; https://github.com/artic-network/fieldbioinformatics) according to expected amplicon lengths, gaining 2 subsets of reads corresponding to the core and polymerase regions. The length-filtered reads were separately mapped to a file containing references of all HBV genotypes using minimap2 (version 2.28) [21]. References starting with the authentic EcoRI-start or with the preCore were used for mapping core or polymerase, respectively. The numbers of primary alignments mapping to the core or polymerase region were counted for each reference. The reference with the highest number of primary alignments was determined for each region and, if identical for both regions, used for consensus sequence generation with the ARTIC pipeline. Length-filtered reads were aligned to the reference, and primers were trimmed. Variants were called with medaka, and a consensus sequence was generated for each region. Generated consensus sequences of the core and polymerase regions were merged if the overlaps were identical. HIV sequencing was performed with a protocol for routine resistance genotyping [22].

Statistical and Bioinformatic Analysis

We compared the demographic and clinical characteristics of participants with HBV alone and HBV/HIV using chi-square and Wilcoxon rank-sum tests. We compared HBeAg by HBV genotype using multivariable logistic regression adjusted for confounders. HBV consensus sequences were aligned with the software Geneious 10.2.6 (RRID:SCR_010519) using MAFFT (PMID: 23329690). For phylogenetic analysis, a tree based on the complete HBV sequence, with references from Genebank, was calculated with the Mr. Bayes plugin [23] using the ngphylogeny pipeline (https://ngphylogeny.fr/). For visualization, the output was exported as a Newick file with support values and visualized with iTol [24]. For calculation of the ratio of nonsynonymous to synonymous substitutions (dN/dS ratio), HBV consensus sequences were separated by genotype, and separate alignments for core and polymerase were generated. These alignments were used for calculation of dN/dS ratio utilizing the SNAP tool from the HIV sequence database (www.hiv.lanl.gov). Resistance-associated mutations for HBV and HIV (if applicable) and immune escape mutations in HBsAg were analyzed with Geno2pheno. We evaluated for recombinants using SimPlot graphs and the phi(w) statistical test [25]. All sequence data are publicly available at Zenodo under https://doi.org/10.5281/zenodo.17396629.

RESULTS

During 2013–2024, 840 HBsAg-positive adults enrolled in the cohort. We excluded from analysis 40 for prior treatment of HBV alone, 178 for unknown or undetectable (<10 IU/mL) HBV DNA levels pretreatment, 274 for insufficient stored samples, and 95 for low HBV DNA levels (between 10 and 500 IU/mL). Aliquots of plasma/serum from the remaining 253 were processed for WGS, and 215 (85.0%) high-quality sequences were generated. In addition to having higher HBV DNA levels, participants whose sequences were included in the final analysis had similar demographics (age and sex, both P > .05) to the overall cohort but were less likely to be HIV negative (32.7% vs 60.3%) and to have higher ALT (median, 31 vs 26; P < .001). The median HBV DNA in the analysis cohort (interquartile range [IQR]) was 4.94 (3.54–7.05) log₁₀ IU/mL, and 77 (39.7%) were HBeAg positive by serology. Seventy-five (34.9%) participants had HBV/HIV, and in that subgroup, the median CD4 count (IQR) was 133 (65–244) cells/mm [3]. None of the participants tested positive for hepatitis delta antibodies. While age and sex were similar, people with HBV/HIV had higher HBV DNA than those with HBV alone (6.5 vs 4.1 log₁₀ IU/mL; P < .001) (Table 1).

Table 1.

Demographic and Pretreatment Clinical Characteristics of Participants With HBV Infection Analyzed by Whole-Genome Sequencing

	Overall (n = 215)	HBV Alone (n = 140)	HBV/HIV (n = 75)	P Valuea
Age, y	33 (27–38)	31 (25–38)	33 (28–38)	.11
Sex				.44
Women	73 (34.0)	45 (32.1)	28 (37.3)
Men	142 (66.0)	95 (67.9)	47 (62.7)
HBV DNA, log10 IU/mL	4.9 (3.5–7.1)	4.1 (3.4–6.0)	6.5 (4.5–8.2)	<.001
Hepatitis B e antigen				<.001
Positive	77 (35.8)	34 (24.3)	43 (57.3)
Negative	117 (54.4)	89 (63.6)	28 (37.3)
Missing	21 (9.8)	17 (12.1)	4 (5.3)
CD4 count, cells/mm3	—	—	133 (65–244)	—
Phase of HBV infection				.17
Acute	12 (5.6)	10 (7.1)	2 (2.7)
Chronic	203 (94.4)	130 (92.9)	73 (97.3)

All values are No. (%) or median (interquartile range).

Abbreviation: HBV, hepatitis B virus.

^aComparison between chronic HBV alone and chronic HBV/HIV based on Wilcoxon rank-sum test for continuous values and chi-square test for categorical ones.

Within the analysis cohort, 12 individuals had acute HBV (including 2 with HBV/HIV) that resolved within 1 year of follow-up. Among the 75 participants with HBV/HIV, 5 evolved to clear HBsAg within 2 years of ART. WGS of HBV revealed 12 sequences with putative immune escape mutations in the HBsAg (6 in gtA and 6 in gtE) and 2 (0.9%) participants with lamivudine drug resistance mutations (Supplementary Table 1). Among the 75 participants with HBV/HIV, 53 had sufficient material to also undertake HIV sequencing. Sequencing of the polymerase/protease was successful for 42 (79.2%) samples, and sequencing of the integrase was successful in 45 (84.9%) samples. All sequenced HIV viruses were clade C. Among these, 16 participants had substitutions in the polymerase and 2 had substitutions in the integrase, which have been associated with reduced susceptibility to antiretroviral treatment (Supplementary Table 1). No resistance-associated substitutions were detected in the protease region. HBV recombinants were absent from the data set based on examination of SimPlots (Supplementary Figure 1B–D) and in the statistical evaluation of both gtE (P = .997) and gtA (P = .571).

Phylogenetic Analysis of HBV Genome Sequences

Figure 1 presents a detailed phylogenetic tree illustrating HBV genotypes, HIV coinfections, acute HBV infections, and cases of HBsAg loss following ART initiation. Two HBV genotypes were detected with similar prevalence: 114 sequences (53.0%) were gtE, while 101 (47.0%) were gtA, all belonging to subgenotype A1. The distribution of HBV genotypes was not statistically different between individuals with and without HIV. Among the 75 people with HBV/HIV, 45 (60%) had gtE and 30 (40%) had gtA. There was no apparent phylogenetic clustering of HBV sequences from individuals with HBV/HIV to suggest different HBV transmission features from counterparts with HBV alone. Interestingly, 11 of 12 (91.7%) acute infections analyzed were gtE. With 2 exceptions, sequences from acute infection were dispersed throughout the phylogenetic tree. In 2 cases, viruses from acute infections clustered closely together. Among the 5 individuals with HBV/HIV who achieved HBsAg loss following antiviral therapy initiation, 3 had gtE and 2 had gtA.

Figure 1.

Phylogenetic tree with HBV sequences from Zambia. Sequences from whole HBV genomes were aligned together with genotype reference sequences and the reported genotype A/E isolate (AB194949) [26] using MAFFT. The phylogenetic tree was calculated with Mr. Bayes using the ng-phylogeny pipeline (https://ngphylogeny.fr/). Genotypes are color-coded as indicated, and infection status is marked in squares outside the sequence name. Abbreviation: HBV, hepatitis B virus.

HBeAg Status by Genotype, Age, and HIV Status

HBeAg serological testing results were available for 194 (90.2%) participants. HBeAg positivity was more frequent in participants with gtA (41.6%) than in those with gtE (30.7%), though the difference was not statistically significant (P = .0778) (Figure 2A). HIV had a significant influence on HBeAg status. In gtA, HBeAg positivity was observed in 24 of 63 (38.1%) people with HBV alone, compared with 18 of 27 (66.7%) in counterparts with HBV/HIV (P = .0204). In gtE, 10 of 60 (16.7%) with HBV alone were HBeAg positive, compared with 25 of 44 (56.8%) with HBV/HIV (P < .0001) (Figure 2B). In HBV alone, HBeAg-positive individuals were significantly younger than their HBeAg-negative counterparts (P = .0362) (Figure 2C). However, this was not seen in HBV/HIV, where people with HBeAg positivity were significantly older than those with HBV alone (P = .0194) (Figure 2D). In HBV/HIV, there was a nonsignificant trend toward lower CD4 T-cell counts in HBeAg-positive individuals (P = .0913) (Figure 2E).

Figure 2.

HBeAg serostatus of the Zambian cohort. A, HBeAg serostatus in gtA and gtE. B&C, HBeAg serostatus in individuals with HBV alone (HBV) and individuals with HBV/HIV in genotype A (B) and genotype E (C). D, Age distribution of HBeAg-positive and -negative patients with HBV alone (HBV) or HBV/HIV. E, CD4 T-cell count in cell/mm³ in HBeAg-positive and -negative patients. P values were calculated by Fisher exact test (A–C) or 1-way ANOVA (D + E). Abbreviations: ANOVA, analysis of variance; gtA, genotype A; gtE, genotype B; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.

Precore Variants in HBV gtE vs gtA

We next analyzed the frequencies of substitutions in the BCP and precore region that impair HBeAg production [27]. This analysis focused on 2 key BCP substitutions (A1762T + G1764A), a variant precore start codon (ATG to any variant), and a substitution introducing a stop codon at precore codon 28 (G1896A) (Figure 3A). BCP/precore variants were detected in 40% of all gtA sequences and 51% of all gtE sequences. Although the overall prevalence of BCP variants (A1762T + G1764A) was comparable between gtA and gtE, significant differences were observed in the frequencies of other variants. The variant precore start codon (ATG variant) was exclusive to gtA, detected in 12.2% of cases alone, but was absent in gtE. In contrast, the precore stop codon (G1896A) was the most frequent variant in gtE, occurring in 22.1% of cases alone and in 8.7% alongside BCP mutations, but it was only detected in 1 patient with gtA. Of note, this patient also had the compensatory C1858T mutation that is required for the G1896A [28]. Consistent with findings on HBeAg serostatus, HBV/HIV was associated with significantly higher frequency of isolates with the prototypic PC/BCP sequence than HBV alone in both gtA (P = .0105) (Figure 3B) and gtE (P = .0052) (Figure 3C).

Figure 3.

Frequency of BCP and precore variants. A, Frequency of the different BCP and precore variants in the cohort. pt = prototype (none of the following substitutions); ATG variant = any variation from ATG; A1762T/G1764A = BCP variant; G1896A = stop codon at W28; A1762T/G1764A/G1896A = BCP variant combined with G1896A. B&C, Frequency of any BCP/precore variant in patients with HBV alone (HBV) or HBV/HIV in genotype A (B) and genotype E (C). P values were calculated by Fisher exact test. Abbreviations: BCP, basal core promoter; HBV, hepatitis B virus.

Distinct Substitution Frequencies in gtA and gtE With HBV/HIV

The median genetic distance for full-length sequences was higher for gtA than gtE (0.012 vs 0.008; P < .001). For the precore/core region (“core”; nt1814-2458), this was 0.014 for gtA vs 0.013 for gtE. For the polymerase (“pol”; nt2307-1623) region, it was 0.012 for gtA and 0.005 for gtE (Figure 4A). The dN/dS ratio was significantly higher in the core region of gtE compared with its pol region (P < .0001) (Figure 4B). Moreover, the dN/dS ratio in the gtE core region was also significantly higher than in the gtA core region (P < .0001) (Figure 4B). Although dN/dS ratios were generally lower in gtA than in gtE, they were significantly reduced in the core region for both genotypes in individuals with HBV/HIV compared with those with HBV alone (gtA: P = .0059; gtE: P = .0286) (Figure 4C, D). Notably, no significant differences were observed in dN/dS ratios within the polymerase (pol) region between HBV alone and HIV/HBV (Figure 4E, F). While no significant correlation was found between CD4 counts and core dN/dS ratios (Figure 4G, H), a trend was seen for gtE viruses (Figure 4H). Higher HBV DNA levels were associated with lower dN/dS ratios for both genotypes (Figure 4I, J). With 1 exception, all individuals with acute infection had gtE. In most cases, sequences from acute infections exhibited low dN/dS ratios (Figure 4C, D). Notably, individuals with HBV/HIV who achieved HBsAg loss (Figure 4D, F) also displayed low dN/dS ratios.

Figure 4.

Genetic distance and substitution frequencies in HBV. A, Genetic distance from the genotype-specific consensus sequence in the precure/core region (“core”; nt1814-2458) and polymerase region (“pol”; nt2307-1623) gene. P values were calculated by 1-way ANOVA with Tukey's multiple comparison. B, Ratio of nonsynonymous to synonymous substitutions (dN/dS ratio) compared with a genotype-specific consensus sequence in the precore (core) and pol ORF in all patients. C, dN/dS ratio in the core ORF of individuals with genotype A with chronic HBV alone, HBV/HIV, acute infection (acute), or FC. D, dN/dS ratio in the core ORF of individuals with genotype E. E, dN/dS ratio in the pol ORF of individuals with genotype A. F, dN/dS ratio in the pol ORF of individuals with genotype E. P values were calculated by Mann-Whitney t test. G, Correlation of dN/dS ratios and CD4 T-cell count in HBV/HIV-coinfected individuals with genotype A. H, Correlation of dN/dS ratios and CD4 T-cell count in HBV/HIV-coinfected individuals with genotype E. I, Correlation of dN/dS ratios with HBV DNA levels in gtA. J, Correlation of dN/dS ratios with HBV DNA levels in gtE. P values and correlation coefficient were calculated by simple linear regression. Abbreviations: ANOVA, analysis of variance; dN/dS, nonsynonymous to synonymous; FC, functional cure; HBV, hepatitis B virus; ORF, open reading frame; pol, polymerase.

DISCUSSION

In a sentinel HBV cohort in Zambia, we used WGS to analyze HBV sequence diversity and substitution frequencies in the context of chronic and acute infection with and without HIV coinfection–induced immune suppression. HBV gtE had a significantly different profile from gtA, including a higher rate of variants leading to HBeAg-negative infection. gtE was also predominant in acute infection. We also found dN/dS ratios that were higher in the core region in gtE compared with gtA, providing evidence for stronger selection pressure on this region. HBV/HIV was associated with a much higher proportion of prototype sequences in the BCP and precore region vs HBV alone. In line with impairment of immune selection pressure, dN/dS ratios were lower in HBV/HIV compared with HBV alone. Interestingly, low dN/dS ratios were also observed in the context of acute resolving HBV and HBsAg loss during tenofovir-treated chronic HBV/HIV. Together these data shed new light on HBV gtE, a prevalent but neglected genotype in Africa, further evidence that immune pressure drives HBV viral evolution, and highlight the value of WGS in viral elimination.

In this study, HBV gtE exhibited lower overall genetic distance in phylogenetic analysis, consistent with its relatively recent emergence in humans and corroborated by findings from smaller studies [8, 29, 30]. We built on past understanding through comparative analysis of dN/dS ratios in gtA vs gtE, revealing 2 key findings: (1) dN/dS ratios were overall higher in gtE than in gtA, indicating that gtE was under greater selection pressure; and (2) among gtE viruses, dN/dS ratios were higher in the core region than in pol, suggesting stronger selective pressure on the core. This suggested a more dynamic evolutionary process and faster adaptation kinetics in gtE than in gtA, potentially due to its relatively recent emergence and subsequent adaptation to the human population. Our observations also aligned with the hypothesis that HBV gtE core adapts more rapidly than pol to selection pressure in humans [31].

We also described HBV sequences in people with HBV/HIV, building on our past description of higher HBV DNA levels and more HBeAg positivity in this group [32]. In this study, viral WGS revealed similar distribution of genotypes in HBV/HIV and HBV alone, which conflicted with other studies where people with HIV had unique HBV transmission patterns [33]. HBV/HIV was also associated with strikingly lower dN/dS ratios in the HBV core region, suggesting altered HBV substitution dynamics in the context of immune suppression. Collectively, higher rates of HBeAg seropositivity, lower prevalence of BCP and precore variants, and reduced dN/dS ratios provided evidence of impaired immune pressure on HBV in HBV/HIV. HBV may revert to a prototype virus in the context of HIV.

We also described the frequency of common mutations associated with HBeAg seroconversion, which is thought to occur earlier in life in Africa than Asia. gtE had multiple variants leading to HBeAg-negative infection, including the G1896A, which was previously reported to be rare in gtE compared with other genotypes [8]. The same G1896A was associated with reduced HBsAg seroclearance in Côte d’Ivoire [15]. We also described in Zambia that compared with gtA, gtE had lower prevalence of HBeAg positivity, seen in only ∼15% of people with HBV alone. This conflicted with a review by Kramvis suggesting that early life loss of HBeAg in Africa was more attributed to gtA than gtE [34] and a report from West Africa suggesting that gtE had lower genetic variability, including HBeAg loss, compared with gtA [30, 33]. Supporting our findings was a report by the Hepatitis B in Africa Collaborative Network, where only 5.2% of adults with chronic HBV alone were HBeAg positive in West Africa, the region where gtE is most prevalent, compared with 17.8% in Southern Africa, where gtA is more prevalent [35 ]. Our results may differ from past reports because of the larger sample size, direct comparison of gtE and gtA in a population with a similar genetic background, and/or due to our use of WGS.

This report also supported the dN/dS ratio as a predictor of clinical outcomes including HBsAg seroclearance, which is the focus of the HBV cure research agenda. Decreasing HBV DNA levels were associated with higher dN/dS ratios, which may suggest that nonsynonymous substitutions have fitness costs to viral replication or are a sign of stronger immune pressure. Previous studies reported that the HBV core protein has the highest degree of adaptation to HLA class I–associated immune pressure by CD8 T cells [31]. This may drive high dN/dS ratios in the core region, something that is reduced by HIV-mediated immunodeficiency.

We also linked low dN/dS ratios to HBsAg loss in acute resolving infection and in people with chronic HBV/HIV who achieved this outcome during ART. As in HBV/HIV, participants with acute HBV had low viral diversity, which was previously described at the nucleotide level [36]. Low viral diversity may reflect the short duration in which the virus was exposed to immune pressure or that variants with multiple substitutions are less likely to be transmitted. The Zambian cohort has reported a relatively high rate of HBsAg seroclearance in chronic HBV/HIV [19], and pretreatment viruses from 5 of these individuals had low dN/dS ratios and low genetic distance from the consensus sequences. This finding is supported by a previous study where low viral diversity at the quasispecies level, based on low haplotype number, predicted HBsAg seroclearance during tenofovir treatment of HBeAg-positive genotype A and genotype D infections [37]. BCP and precore variants have been associated with reduced HBsAg seroclearance [38]. While the phenomenon of HBsAg seroclearance during ART-treated HBV/HIV is thought to be mediated by restoration of immunity (due to HIV RNA suppression), less is known about HBV viral aspects. When tenofovir-based ART is initiated, “unadapted” HBV with no or few substitutions may be more susceptible to the combined mechanisms of direct inhibition of reverse transcriptase by tenofovir and immune reconstitution, which can sometimes trigger ALT flares. In relation to acute resolving HBV, our findings of low dN/dS ratios are in line with past analyses, where deep sequencing of the surface region [39] and WGS analysis of variants from the quasispecies [40] revealed lower diversity in acute than in chronic HBV.

This work also shed light on HBV transmission in Africa, finding that nearly every acute HBV infection was gtE, unlike among chronic infections. Although the number of acute infections was too low for solid conclusions, gtE may be more common in recent adult infections. In India, genotypic differences between acute and chronic HBV were also described, with gtD being more associated with acute sexual transmission [41]. Unlike the comparison between acute and chronic infection, we did not see a statistical difference in HBV genotype between people with and without HIV. This conflicts with a report from Cameroon, where HBV gtA was more common than gtE in those with HIV [30]. Our data suggest that HBV and HIV are transmitted independently in Zambia; most people with and without HIV likely acquired HBV at a similar time (ie, primarily at birth or early childhood).

These data are important to the field for several reasons. First, they reveal that gtE in Africa may have unique transmission and highlight clinical aspects that warrant further understanding in the context of HBV elimination. Better understanding is needed, for example, on whether gtE is more easily transmitted from mother to child or sexually in the adult population, compared with other genotypes. High rates of precore variants in gtE may explain why CHB among adults in Africa is predominantly HBeAg negative. We did not find evidence that HBV transmission differs in people with HIV compared with those without it. Higher prevalence of HBV in people with HIV in Zambia might be instead due to viral re-activation. We also linked the dN/dS ratio to resolution of infection, raising this as a possible viral biomarker, which could play a role in clinical staging of CHB and should be further evaluated as a predictor of functional cure. Viral sequencing capacity has grown substantially since the COVID pandemic, and this analysis supports an expanded role for WGS. Albeit rare, HBV drug resistance and immune escape mutations were also detected in the Zambia cohort. It is important for the Africa region to have surveillance of mutations like these that could threaten viral elimination in the future.

This analysis had significant strengths, including access to a unique and relatively large cohort in Africa and the use of WGS, but also several limitations. One limitation was that some cohort participants did not end up in the final analysis due to insufficient samples or low levels of viremia. The minimum of 500 IU/mL HBV-DNA was selected to allow efficient sequencing and generation of high-quality WGS data. It is possible that specific HBV genotypes associate with distinct immune control and low viral load. In this case, focusing on samples with HBV-DNA concentrations above a certain threshold could have introduced a bias into the analysis of genotype distribution. Moreover, our analysis of genetic distance was limited by its cross-sectional nature and reliance on genotype-specific reference consensus sequences to estimate substitution rates. Determining precise substitution rates over time and identifying residues under selection pressure would require longitudinal viral sequencing, which is not feasible after antiviral therapy initiation. Additionally, host HLA types were unavailable, preventing direct linkage of specific substitutions to CD8 and CD4 T-cell immunity. Finally, the data presented in this paper come from a single-country, single-cohort population, limiting generalizability.

In summary, HBV WGS within a unique Zambian HBV cohort yielded new information on circulating HBV genotypes, transmission, and viral evolution under immune pressure.

Supplementary Data

Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.