Lamivudine/Adefovir Treatment Increases the Rate of Spontaneous Mutation of Hepatitis B Virus in Patients

Abstract

The high levels of genetic diversity shown by hepatitis B virus (HBV) are commonly attributed to the low fidelity of its polymerase. However, the rate of spontaneous mutation of human HBV in vivo is currently unknown. Here, based on the evolutionary principle that the population frequency of lethal mutations equals the rate at which they are produced, we have estimated the mutation rate of HBV in vivo by scoring premature stop codons in 621 publicly available, full-length, molecular clone sequences derived from patients. This yielded an estimate of 8.7 × 10-5 spontaneous mutations per nucleotide per cell infection in untreated patients, which should be taken as an upper limit estimate because PCR errors and/or lack of effective lethality may inflate observed mutation frequencies. We found that, in patients undergoing lamivudine/adefovir treatment, the HBV mutation rate was elevated by more than sixfold, revealing a mutagenic effect of this treatment. Genome-wide analysis of single-nucleotide polymorphisms indicated that lamivudine/adefovir treatment increases the fraction of A/T-to-G/C base substitutions, consistent with recent work showing similar effects of lamivudine in cellular DNA. Based on these data, the rate at which HBV produces new genetic variants in treated patients is similar to or even higher than in RNA viruses.

Fig 1. Premature stop-codon mutations found in HBV gene C.

Sequence labels indicate the GenBank accession and the patient code according to Table 1. Below is shown the consensus sequence and the colored sequence logo summarizes variability at each position. Only variable sites are shown in the patient sequences, and stop codons are highlighted in red. As also indicated on Table 2, some stop codons appeared repeatedly in the same patient, or even in different patients. Similar alignments could be obtained for the other HBV genes by retrieving GenBank accession numbers from S1 Table.

Fig 2. Phylogenetic analysis of HBV sequences used in this study.

A neighbor-joining containing the 621 full-length sequences used is shown. Patient names are as in Table 1. Sequences from untreated patients are marked in blue and those from treated patients in red. For each indicated patient, numbers show the bootstrap value of the node that delimitates the sequences from this patient. Sequences from patients N16 to N22 correspond to a study in which several family members were studied and are partially intermingled (not shown). Patients N13 and N14 are not shown because their sequences did not form well-defined monophyletic groups. Letters in the center of the tree indicate the viral genotype.

Fig 3. HBV mutational spectrum in untreated patients and in patients undergoing lamivudine/adefovir treatment.

The mutational spectrum was obtained in sequences derived from untreated (blue) or treated (red) patients by scoring all intra-patient SNPs along the HBV genome. Box plots indicate the relative contribution of each substitution type to the total SNPs found. Lower and upper box limits indicate percentiles 25^th and 75^th, respectively, and the middle line shows the median. Whiskers show the 10^th and 90^th percentiles, and outlying points are plotted individually. Differences between treated and untreated patients in the frequency of each substitution type were assessed by a Mann-Whitney non-parametric test (***: p < 0.001; **: 0.001 < p < 0.01; *: 0.01 < p < 0.05; ns: non-significant).