Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

The profile of mutational clusters associated with lamivudine resistance can be constrained by HBV genotypes.

BACKGROUND/AIMS: To investigate the different clusters of mutations associated with lamivudine resistance in HBV genotypes D and A. METHODS: HBV reverse transcriptase sequences of 89 HBV-infected patients failing lamivudine treatment were analyzed. The association of mutations with HBV genotypes was assessed by Chi-Squared test and multivariate logistic regression analysis. Covariate analysis was based on hierarchical clustering. RESULTS: In genotype A, the rtM204V (prevalence: 68.2%) was the main sign of lamivudine failure. Multivariate analysis confirmed that genotype A is the only predictor for rtM204V emergence (OR: 14.5 [95% CI: 1.3-158], P=0.02). Covariate analysis showed that rtM204V clusters with rtL180M, rtL229V (corresponding to sF220L in the HBsAg), and, interestingly, with HBsAg mutation sS207N (bootstrap=0.95). Both sF220L and sS207N co-localized in the fourth transmembrane HBsAg domain. In contrast, in genotype D the primary mutations rtM204V and rtM204I occurred with similar prevalence (39.1% versus 45.3%, P=0.47), and showed a distinct pattern of compensatory mutations. rtM204V clusters with mutations localized in the RT-B domain (rtV173L, rtL180M, and rtT184A/S) (bootstrap=0.94), while rtM204I clusters with mutations localized in the RT-A domain (rtS53N, rtT54Y, and rtL80I/V) (bootstrap=0.96) (without associations with HBsAg specific mutations). CONCLUSIONS: HBV genotype plays an important role in driving RT evolution under lamivudine treatment, and thus can be relevant for therapeutic sequencing, immunological response and disease progression.

Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection.

BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.

Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection.

Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0-6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001). Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate). In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.

Lamivudine-resistance mutations can be selected even at very low levels of hepatitis B viraemia.

OBJECTIVE: To investigate lamivudine (LAM)-resistance profiles of hepatitis B virus (HBV) at the early stages of virological breakthrough (serum HBV-DNA 12-345IU/ml) or when HBV-DNA is undetectable. METHODS: Sixty-four HBV-mono-infected patients were enrolled: 25 had virological breakthrough with serum HBV-DNA ranging from 12 to 345IU/ml during first-line LAM-monotherapy; 24 were on LAM-monotherapy, and 15 were on LAM+adefovir dipivoxil (ADV) with undetectable serum HBV-DNA (<12IU/ml). RESULTS: HBV-reverse transcriptase was successfully sequenced in 22 (88.0%) LAM-treated patients with HBV-DNA between 12 and 345IU/ml, and in 12 (30.8%) patients receiving LAM (±ADV) with HBV-DNA<12IU/ml. Drug-resistance mutations were observed in 17 (77.2%) LAM-treated patients with virological breakthrough: 8 M204V, 7 M204I, 1 M204I/V, and 1 A181T. One or ≥2 compensatory mutations were found in 10 (58.8%) and in 4 (23.5%) patients. Drug-resistance mutations were present also in patients with undetectable serum HBV-DNA: M204I was detected in 2 patients receiving LAM-monotherapy, and V84M in 1 patient receiving LAM+ADV. CONCLUSION: Overall findings support the existence of drug-resistance mutations even at very low levels of viral replication. The persistence of low-level HBV replication and consequent drug-resistance emergence should be considered when choosing therapeutic strategies.