A. Spanò

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.

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.

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.

Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen

INTRODUCTION The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. METHODS 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. RESULTS Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. CONCLUSIONS Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.

Characterization of drug-resistance mutations in HBV D-genotype chronically infected patients, naïve to antiviral drugs.

Presence of drug-resistance mutations in drug-naïve hepatitis B virus (HBV) infected patients can seriously compromise response to antiviral treatment. Therefore, our study was aimed at defining the prevalence of HBV drug-resistance in a population of 140 patients, all infected with HBV-D-genotype (the most common HBV-genotype in Eastern Europe, Mediterranean countries and Middle East) and naïve to antiviral therapy. HBV reverse-transcriptase (RT) region was sequenced and analyzed for 20 mutations, confirmed by in vitro studies as associated with resistance to nucleos(t)ide HBV-RT inhibitors (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V). Amino acid changes at other six RT positions, potentially associated with resistance, were also analyzed (rtV84M-rtV191I-rtV207L-rtV214A-rtQ215S-rtI233V). Overall, only 2/140 (1.4%) patients carried primary drug-resistance mutations [rtA181V (0.7%), and rtA194T (0.7%)], while 3/140 (2.1%) patients harbored the secondary mutations rtV173L (1.4%) and rtL180M (0.7%). Additionally, five polymorphic mutations, with a suggested role in drug resistance, were detected [rtQ215S (12.8%), rtI233V (4.3%), rtV214A (3.6%), rtV191I (0.7%), rtV207L (0.7%)]. Notably, no YMDD mutations, namely rtM204V/I, were found. Taken together, the rate of important drug resistance mutations in naïve HBV D-genotype infected patients is today very low, and suggests the potential full efficacy of new-generation antiviral drugs used in first line therapy. Whether such low rate can be extrapolated to non HBV-D subtypes, requires a detailed investigation to be performed in a different cohort of patients.

The synergistic interaction of interferon types I and II leads to marked reduction in severe acute respiratory syndrome-associated coronavirus replication and increase in the expression of mRNAs for interferon-induced proteins.

Interferon (IFN)-α, -β and -γ have been shown to be only marginally effective against severe acute respiratory syndrome coronavirus (SARS-CoV) replication in Vero cell lines. We investigated the combination of type I IFNs (IFN-α or -β) and IFN-γ for antiviral activity and found that such combinations synergistically inhibited SARS-CoV replication in Vero cells, using yield reduction assay and the isobologram and combination index methods of Chou and Talalay for evaluation. The highly synergistic anti-SARS-CoV action of type I IFNs and IFN-γ parallels the marked increase in 2′-5′-oligoadenylate synthetase and p56 mRNAs following exposure in Vero cells to either IFN-α or -β and IFN-γ compared with the transcriptional levels obtained after stimulation with either IFN alone. These results demonstrate that SARS-CoV, although only moderately sensitive to the antiviral action of the individual types of IFN, is highly sensitive to a combination of type I and II IFNs, which suggests that such combinations may have potential in the treatment of SARS-CoV infections.

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.

Intra-hepatic messenger RNA levels for interferons and related genes in hepatitis C virus/HIV co-infected patients.

Intra-hepatic levels of mRNA for IFN-a, IFN-y, IFN type 1 receptor (IFNAR-1) and PKR were determined in hepatitis C virus (HCV)/HIV co-infected and HCV mono-infected patients. In co-infected patients, IFN-a mRNA was upregulated and correlated with HIV-1 viraemia. IFN-y, IF-NAR-1 and PKR mRNA were detected in mono-infected, but not in co-infected patients. These findings suggest that in HCV/HIV co-infected individuals the ability to respond to IFN-a is impaired, probably because of the absence of its receptor.

Appearance of HbeAg in an Occult Persistent Hepatitis B Virus Infection

Occult hepatitis B virus infection (OBI) is characterized by the presence of ongoing viral replication with very low levels of viremia (<200 IU/ml), and negativity for HBsAg, while the so-called ‘false’ OBI with higher levels of HBV-DNA that are negative for HBsAg are usually due to the occurrence of mutations of the HBsAg sequence that may alter the recognition by some immunoassays. We describe here a case of occult HBV infection that combines both aspects. A male patient with severe systemic diseases, positive for anti-HBc and anti-HBs and negative for all other HBV markers, including HBsAg, since at least 4 years, showed a positivity for HBeAg at a follow-up control in November 2008; HBV-DNA testing by real-time PCR evidenced very low levels of viremia (<40 IU/ml), direct sequencing of the surface antigen-coding and Pol/RT coding regions allowed the identification of genotype D, serotype adw2, one immune escape mutation (G145R) and no drug resistance mutations. The positivity for HBeAg could be attributed to a superinfection in a naturally immune subject or to reactivation of a latent infection; the mutated virus had a reduced fitness and was therefore able to replicate only at low levels, resulting in a mild form of occult HBV infection.

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.