Substitution Rate of the Hepatitis B Virus Surface Gene
Substitution Rate of the Hepatitis B Virus Surface Gene
Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10 to 10 cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0-8 per patient), resulting in an average substitution rate of 5.1 × 10 nucleotide changes/site/year (range: 0-1.3 × 10). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA ≥10 cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 × 10), while in the 10 patients with viremia below 10 cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 × 10). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.
Hepatitis B virus (HBV) has a DNA genome, but it replicates via an RNA intermediate. The substitution rate of HBV is thus expected to be determined by the low fidelity of its reverse transcriptase. However, more than half of the HBV genome consists of overlapping, frame shifted reading frames: the gene for the surface (S) protein completely overlaps with the polymerase gene, while the genes for the core, polymerase, and X-proteins are partially overlapping. Overlapping genes and secondary structures of the HBV genome involved in regulation of replication, impose a constraint on the number and nature of substitutions occurring in the HBV genome. The evolution of HBV is thus proposed to be an example of 'constrained viral evolution'.
Few studies on the substitution rate of HBV are available, probably because blood samples, spanning many years of HBV infection are rare. Some studies present long-term substitution rates based on the analysis of HBV-infected mother-child sample pairs and the theoretical construction of a common ancestor HBV. Other studies present substitution rates based on a comparison of cross-sectional samples to a consensus sequence. Reported HBV substitution rates should thus be interpreted and compared with caution.
The prevalence of hepatitis B virus infection in North-west Europe is low, for example in the Netherlands the prevalence of chronic HBV infection is estimated to be 0.4%. Because of this low prevalence, no universal childhood vaccination against HBV has been implemented in some European countries, instead HBV-vaccination programs targeted at children and adults with increased risk of HBV infection have been introduced. Knowledge of HBV transmission among and between the at-risk groups is necessary to decide whether such targeted vaccination programs are justified. To evaluate the impact of the different HBV vaccination programs in the Netherlands, serum of persons with acute hepatitis B is collected and the HBV S-gene sequence is determined. About half of the recovered HBV isolates belong to the HBV genotype A lineage and 55% (56/101) of these isolates have identical S-gene sequences, see the phylogenetic tree in reference. The interpretation of these HBV sequence data is hampered by our limited knowledge of the substitution rate of HBV in persistently and acutely infected persons, and of factors that may influence the substitution rate.
Recently, Osiowy et al. described the substitution rate of HBV in six low viremic, HBV e-antigen negative, precore stop-codon positive HBV carriers, by analysing six pairs of blood samples spanning an interval of 25 years. Here we report the substitution rate in the HBV surface gene, as observed in pairs of blood samples of 40 untreated, chronically HBV-infected patients, spanning 210 cumulative years of follow-up. The 40 patients include e-antigen positive and negative patients; with HBV DNA levels ranging from 10 to 10 cps/mL; belonging to the five major HBV genotypes (A, B, C, D, and E).
Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10 to 10 cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0-8 per patient), resulting in an average substitution rate of 5.1 × 10 nucleotide changes/site/year (range: 0-1.3 × 10). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA ≥10 cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 × 10), while in the 10 patients with viremia below 10 cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 × 10). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.
Hepatitis B virus (HBV) has a DNA genome, but it replicates via an RNA intermediate. The substitution rate of HBV is thus expected to be determined by the low fidelity of its reverse transcriptase. However, more than half of the HBV genome consists of overlapping, frame shifted reading frames: the gene for the surface (S) protein completely overlaps with the polymerase gene, while the genes for the core, polymerase, and X-proteins are partially overlapping. Overlapping genes and secondary structures of the HBV genome involved in regulation of replication, impose a constraint on the number and nature of substitutions occurring in the HBV genome. The evolution of HBV is thus proposed to be an example of 'constrained viral evolution'.
Few studies on the substitution rate of HBV are available, probably because blood samples, spanning many years of HBV infection are rare. Some studies present long-term substitution rates based on the analysis of HBV-infected mother-child sample pairs and the theoretical construction of a common ancestor HBV. Other studies present substitution rates based on a comparison of cross-sectional samples to a consensus sequence. Reported HBV substitution rates should thus be interpreted and compared with caution.
The prevalence of hepatitis B virus infection in North-west Europe is low, for example in the Netherlands the prevalence of chronic HBV infection is estimated to be 0.4%. Because of this low prevalence, no universal childhood vaccination against HBV has been implemented in some European countries, instead HBV-vaccination programs targeted at children and adults with increased risk of HBV infection have been introduced. Knowledge of HBV transmission among and between the at-risk groups is necessary to decide whether such targeted vaccination programs are justified. To evaluate the impact of the different HBV vaccination programs in the Netherlands, serum of persons with acute hepatitis B is collected and the HBV S-gene sequence is determined. About half of the recovered HBV isolates belong to the HBV genotype A lineage and 55% (56/101) of these isolates have identical S-gene sequences, see the phylogenetic tree in reference. The interpretation of these HBV sequence data is hampered by our limited knowledge of the substitution rate of HBV in persistently and acutely infected persons, and of factors that may influence the substitution rate.
Recently, Osiowy et al. described the substitution rate of HBV in six low viremic, HBV e-antigen negative, precore stop-codon positive HBV carriers, by analysing six pairs of blood samples spanning an interval of 25 years. Here we report the substitution rate in the HBV surface gene, as observed in pairs of blood samples of 40 untreated, chronically HBV-infected patients, spanning 210 cumulative years of follow-up. The 40 patients include e-antigen positive and negative patients; with HBV DNA levels ranging from 10 to 10 cps/mL; belonging to the five major HBV genotypes (A, B, C, D, and E).