Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters

Abstract

Background & aims: We aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS).

Methods: Fifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies.

Results: HBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome.

Conclusions: Higher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome.

Lay summary: HBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

Keywords: ALT, alanine aminotransferase; AUC, area under curve; BCP, basal core promoter; C, core; CHB, chronic hepatitis B; Chronic hepatitis B; EOT, end of treatment; HBeAg seroconversion; IFN, interferon; IFN-NR, IFN-non-responders; IFN-No-eSC, IFN-treated HBeAg non-seroconverters; IFN-RS, IFN-responders; IFN-eSC, IFN-treated HBeAg seroconverters; Intra-host single nucleotide variants; NGS, next-generation sequencing; ORFs, open reading frames; P, polymerase; S, surface; TN-No-eSC, treatment-naïve non-seroconverters; TN-eSC, treatment-naïve HBeAg seroconverters; dN, nonsynonymous substitution rate; dS, synonymous substitution rate; iSNVs, intra-host single-nucleotide variants.

Graphical abstract

Fig. 1

The relationships between nucleotide diversity and the levels of viral load and ALT among the treatment-naïve and IFN-treated CHB patients. (A, B) The relationships between nucleotide diversity and the levels of viral load (A) and ALT (B) among the treatment-naïve CHB patients. (C, D) The relationships between nucleotide diversity and the levels of viral load (C) and ALT (D) among the IFN-treated patients. The relationship and significance are determined by Spearman correlation. ALT, alanine aminotransferase; CHB, chronic hepatitis B; IFN, interferon.

Fig. 2

The dynamic changes of HBV nucleotide and amino acid diversities along with spontaneous HBeAg seroconversion. (A) and (B) represent the nucleotide diversities of HBV whole genome, C, P, S, and X genes, respectively, before (T1), at (T2), and after (T3) the year of HBeAg seroconversion in TN-eSC (orange) and TN-No-eSC (grey) groups. (C) and (D) represent the amino acid diversities of the non-overlapping regions within HBV whole genome, C, P, S, and X genes, respectively, before (T1), at (T2), and after (T3) the year of HBeAg seroconversion in TN-eSC (orange) and TN-No-eSC (grey) groups. The value is shown as the mean ± SD. Asterisks indicate the statistical significance in comparison between groups by Mann-Whitney U test (∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005, ∗∗∗∗p <0.001). C, core; P, polymerase; S, surface; TN-eSC, treatment-naïve HBeAg seroconverters; TN-No-eSC, treatment-naïve non-seroconverters.

Fig. 3

The dynamic changes of HBV nucleotide and amino acid diversities along with IFN treatment. (A) and (B) represent the nucleotide diversities of HBV whole genome, C, P, S, and X ORFs, respectively, before (T1), at 12 weeks of (T2), at the end of (T3), and 24 weeks after (T4) IFN treatment in IFN-RS (green) and IFN-NR (yellow) groups. (C) and (D) represent the amino acid diversities of the non-overlapping regions within the HBV whole genome, C, P, S, and X ORFs, respectively, before (T1), at 12 weeks of (T2), at the end of (T3), and 24 weeks after (T4) IFN treatment in IFN-RS (green) and IFN-NR (yellow) groups. Values are shown as the mean ± SD. Asterisks indicate the statistical significance in comparison between groups by Mann-Whitney U test (∗p <0.05, ∗∗p <0.01). C, core; IFN, interferon; IFN-NR, IFN-non-responders; IFN-RS, IFN-responders; ORFs, open reading frames; P, polymerase; S, surface.

Fig. 4

The dN/dS ratios along with HBeAg seroconversion. (A) The dN/dS ratios of C, P, S, and X ORFs in the treatment-naïve groups. The colours, orange and grey, indicate the TN-eSC and the TN-No-eSC groups, respectively. (B) The dN/dS ratios of C, P, S, and X ORFs in the IFN-treated groups. The colours green and yellow indicate the IFN-RS and the IFN-NR groups, respectively. Values are shown as the median (IQR). Asterisks indicate the statistical significance by Mann-Whitney U test (∗∗∗p <0.005, ∗∗∗∗p <0.001). C, core; dN, nonsynonymous substitution rate; dS, synonymous substitution rate; IFN, interferon; IFN-NR, IFN-non-responders; IFN-RS, IFN-responders; ORFs, open reading frames; P, polymerase; S, surface; TN-eSC, treatment-naïve HBeAg seroconverters; TN-No-eSC, treatment-naïve non-seroconverters.

Fig. 5

The genetic evolutionary rate of HBV within the treatment-naïve and the IFN-treated HBeAg-positive patients. (A) The genetic evolutionary rate of HBV in the treatment-naïve groups. The colours orange and grey indicate the TN-eSC and the TN-No-eSC groups, respectively. (B) The genetic evolutionary rate of HBV in the IFN-treated groups. The colours green and yellow indicate the IFN-RS and the IFN-NR groups, respectively. Values are shown as the mean ± SD. Asterisks indicate the statistical significance by Mann-Whitney U test (∗∗∗p <0.005, ∗∗∗∗p <0.001). IFN, interferon; IFN-NR, IFN-non-responders; IFN-RS, IFN-responders; TN-eSC, treatment-naïve HBeAg seroconverters; TN-No-eSC, treatment-naïve non-seroconverters.

Fig. 6

The levels of iSNVs among the treatment-naïve and the IFN-treated HBeAg-positive CHB patients. (A) The normalised numbers of iSNVs in the HBV whole genome in the treatment-naïve groups. (B) The iSNVs attributes within the non-overlapping region of the HBV genome in the treatment-naïve groups. (C) The normalised numbers of iSNVs in the HBV whole genome in the IFN-treated groups. (D) The iSNVs attributes within the non-overlapping region of the HBV genome in the IFN-treated groups. NS represents the nonsynonymous substitution and S indicates the synonymous substitution. The number of iSNVs is normalised with the sequenced read length. Values are shown as the mean ± SD. The values of SD in panels (B) and (D) were computed by the SD derived from the normalised total iSNVs within the non-overlapping region. Asterisks indicate the statistical significance by Mann-Whitney U test (∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005, ∗∗∗∗p <0.001). CHB, chronic hepatitis B; IFN, interferon; IFN-NR, IFN-non-responders; IFN-RS, IFN-responders; iSNVs, intra-host single-nucleotide variants; TN-eSC, treatment-naïve HBeAg seroconverters; TN-No-eSC, treatment-naïve non-seroconverters.

Fig. 7

The prevalently non-synonymous iSNVs with statistical importance in the non-overlapping regions of HBV genome. The prevalent iSNVs with non-synonymous substitutions and significantly different between (A) the treatment-naïve and (B) the IFN-treated groups. The nucleotide derived from the compiled HBV genotype B sequences is displayed as prefix to the position nucleotide. The nucleotide of iSNV is suffixed at the position of nucleotide. The non-synonymous substitution is presented as the following sequence, protein: dominant, residue location, and mutant. The dash sign indicates the stop codon. The distribution of the mutant percentage, that is the iSNV percentage, is shown as median with lower and upper hinges corresponding to the first and third quartiles on the panels. The statistical significance of difference between groups is calculated by the Mann-Whitney U test. Asterisks indicate the statistical significance (∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005). IFN, interferon; IFN-NR, IFN-non-responders; IFN-RS, IFN-responders; iSNVs, intra-host single-nucleotide variants; TN-eSC, treatment-naïve HBeAg seroconverters; TN-No-eSC, treatment-naïve non-seroconverters.