Patients and samples
We performed direct PCR sequencing and UDPS on 17 plasma samples from 17 NRTI-naive, HBV-infected patients and 20 plasma samples from 11 NRTI-treated, HBV-infected patients. Six of the NRTI-treated patients were HIV-1 infected; 2–3 samples obtained at different times were available for these patients. The median plasma HBV DNA level was 9.9 × 105 IU/mL (range, 2.8 × 104–3.8 × 107 IU/mL) for the samples from NRTI-naive patients and 3.8 × 106 IU/mL (range, 1.1 × 104–2.5 × 108 IU/mL) for the samples from NRTI-treated patients.
Samples from NRTI-naive patients: direct PCR sequencing and UDPS
Of the 17 NTRTI-naive patients, 11 were infected with genotype B HBV strains, 3 with genotype A strains, and 3 with genotype C strains. Direct PCR sequencing detected a mean of 5.9 mutations/sample (range, 0–13 mutations/sample) (). UDPS detected a mean of 4.6 additional mutations/sample (range, 0–14 additional mutations/sample) that were not detected by direct PCR sequencing. Among the NRTI-resistance mutations, M204I was detected in 1.3% of the sequence reads in the sample from subject A7; A181T and M204I were present in 1.0% of the sequence reads in the sample from subject E6. For sample A7, M204I was found in 1 of 64 clones sequenced by dideoxynucleotide sequencing; however, that clone had 2 stop codons and G-to-A hypermutation. For the sample from subject E6, A181T and M204I were each found in 1 of 80 molecular clones; the clone with M204I had G-to-A hypermutation and 2 stop codons.
Mutations in hepatitis B virus (HBV) reverse-transcriptase genes detected by direct polymerase chain reaction (PCR) sequencing and ultra-deep pyrosequencing (UDPS) in nucleoside- and nucleotide-naive HBV-infected patients.
Samples from NRTI-treated patients: direct PCR sequencing and UDPS
Of the 11 NRTI-treated patients, 5 had received lamivudine (3TC) alone; 2 had received 3TC and adefovir (ADV); 2 had received 3TC, ADV, and entecavir (ETV); 1 had received ADV alone; and 1 had received 3TC, tenofovir (TDF), and emtricitabine (FTC). Four patients had had a sample obtained while they were not taking NRTIs for a period of 3–19 months. Three patients were infected with genotype G HBV strains, 3 with genotype A strains, 3 with genotype B strains, 1 with a genotype C strain, and 1 with a genotype D strain.
Direct PCR sequencing detected a mean of 9.9 mutations/sample (range, 0–23 mutations/sample)—including NRTI-resistance mutations—in 16 samples obtained from 9 of 11 patients (). The 4 samples without NRTI-resistance mutations were collected from patients who were not receiving NRTI therapy at the time the sample was obtained. The direct PCR sequences were dominated by 3TC-resistance mutations: L180M and M204V/I in 9 samples; V173L, L180M, and M204V in 5 samples; L180M, A181V (a mutation associated with ADV and 3TC resistance), and M204V in 1 sample; and V173L alone in 1 sample.
Table 3 Mutations in hepatitis B virus (HBV) reverse-transcriptase genes detected by direct polymerase chain reaction (PCR) sequencing and by ultra-deep pyrosequencing (UDPS) of 20 samples from 11 patients infected with HBV and treated with nucleoside and/or (more ...)
UDPS identified additional NRTI-resistance mutations in 10 of 20 samples from 5 of 11 NRTI-treated patients. The most common additional mutation was V173L, which was found in 5 samples for which the direct PCR sequence contained L180M and M204V/I. The ETV-resistance mutation T184S was detected at a prevalence of 2%–3% in 2 samples from a 3TC-treated patient for whom the HBV direct PCR sequence contained L180M and M204V. The 3TC-resistance mutation L80V and the ETV-resistance mutation S202G were detected at prevalences of 1.5% and 9.9%, respectively, in a patient who had received 3TC, ADV, and ETV. V173L/M, L180M, A181T, and M204V were detected in a patient who had received 3TC for 52 months but had not been receiving therapy for 27 months at the time the sample was obtained. The ADV-resistance mutation N236T was detected at a prevalence of 6.6% in a patient who had received 3TC and ADV but had not been receiving therapy for 19 months at the time the sample was obtained. Clonal sequencing performed on this sample detected N236T in 2 of 89 clones.
Of the 6 HIV-1–infected patients, 5 subsequently attained plasma HBV DNA levels below the limit of quantification (~60 IU/mL), including 3 patients who received TDF and FTC (subjects 1284, 4089, and 26278), 1 patient who changed to TDF and 3TC (subject 1329), and 1 patient who changed to TDF and ETV (subject 7774). One HIV-1–infected patient treated with TDF and FTC (subject 16375) experienced a decrease in plasma HBV levels from 2 million IU/mL to a persistently detectable level of viremia that has ranged from ~300 to 2000 IU/mL.
Coinfections with genotypes G and A
Eight samples from 3 patients were found to have genotype G sequences by direct PCR sequencing. Five of these samples from 2 patients contributed more than half of the low-prevalence mutations observed among the NRTI-treated patients (mean, 22.2 additional mutations/sequence). The vast majority of these low-prevalence mutations were consensus genotype A variants, a result that is consistent with reports that genotype G viruses usually occur in combination with genotype A viruses [21
]. Limiting dilution clonal sequencing of viruses from 1 genotype G sample (subject 1284-1) demonstrated genotype A viruses in 4 of 36 clones.
G-to-A hypermutation was present in sequence reads for 0.49% (range, 0–2.5%) and 0.66% (range, 0–3.2%) of NRTI-naive and NRTI-treated patients, respectively (P = .5). Among the 6 patients from whom multiple samples were obtained, the percentage of hypermutated sequence reads was somewhat more similar within samples from a single patient than among samples from different patients, which suggests that the proportion of viruses with G-to-A hypermutation may be a property of the infecting virus or host environment (P = .06, by the Fligner-Killeen test of homogeneity of variances). Of the hypermutated sequence reads, 37% had ≥1 stop codons in RT, compared with <1% in the nonhypermutated sequence reads.
shows the influence of G-to-A hypermutation at the 3 codons at which a G-to-A change is responsible for a drug-resistance mutation (A181T, A194T, and M204I). Without filtering out sequence reads that met the criteria for G-to-A hypermutation, 8 of these mutations in samples from NRTI-naive patients and 6 in samples from NRTI-treated patients were detected as minor variants at a prevalence of ≥1.0%. After hypermutated sequence reads were excluded, 3 mutations in samples from NRTI-naive patients and 2 in samples from NRTI-treated patients had a prevalence of ≥1.0%.
Influence of G-to-A hypermutation on the percentage of sequence reads containing the nucleoside and/or nucleotide reverse-transcriptase inhibitor (NRTI)–resistance mutations rtA181T, rtA194T, and rtM204I.
S protein mutations
The direct PCR and UDPS RT sequences were translated in a +1 reading frame to identify mutations of possible significance in the overlapping S protein. shows 13 stop codons present in 10 samples from NRTI-naive patients and 27 stop codons present in 7 samples from NRTI-treated patients. Three of the 13 residues with stop codons were always associated with RT amino acid mutations, of which 2 (rtA181T and rtV191I) have previously been reported [23
Stop codons detected at a prevalence of ≥2% by direct polymerase chain reaction sequencing or ultra-deep pyrosequencing.
Of the mutations reported to be associated with decreased hepatitis B surface antigen immunologic reactivity [14
], only sM133L and sP120T were detected in this study. sM133L was present as part of a mixture with wild type in the direct PCR sequence for 2 NRTI-naive patients and as a minor variant detectable only by UDPS for 2 NRTI-naive patients and 1 NRTI-treated patient. sP120T was detected as a minority variant in 1 NRTI-treated patient.