N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.

Background & Aims: HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients.

Methods: Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group.

Similar evolution of hepatitis B virus quasispecies in patients with incomplete adefovir response receiving tenofovir/emtricitabine combination or tenofovir monotherapy.

Background & Aims: Adefovir (ADV) resistance mutations induce low-level cross-resistance to tenofovir in vitro. Our aim was to compare viral kinetics, nucleos(t)ide analog resistance mutations, and quasispecies (QS) evolution during therapy with tenofovir disoproxil fumarate (TDF) or emtricitabine + TDF (FTC/TDF) in selected patients with incomplete ADV responses.

Methods: Patients with chronic hepatitis B and incomplete response to ADV were randomized in a double-blind trial of TDF vs.

High incidence of treatment-induced and vaccine-escape hepatitis B virus mutants among human immunodeficiency virus/hepatitis B-infected patients.

Unlabelled: Anti-hepatitis B virus (HBV) nucleos(t)ides analogs (NA) exert selective pressures on polymerase (pol) and surface (S) genes, inducing treatment resistance and increasing the risk of vaccine escape mutants. The rate of emergence for these mutations is largely unknown in patients coinfected with human immunodeficiency virus (HIV) and HBV undergoing dual-active therapy. In a 3-year, repeat-sampling, prospective cohort study, HBV viral genome sequences of 171 HIV-HBV coinfected patients, presenting with HBV viremia for at least one visit, were analyzed every 12 months via DNA chip.

Decreased infectivity of nucleoside analogs-resistant hepatitis B virus mutants.

Background & Aims: To understand the mechanisms of emergence and selection of HBV polymerase variants, which may also harbor mutations in the overlapping envelope protein, we analyzed the in vitro virus production and infectivity of the main viral mutants resistant to lamivudine and adefovir.

Methods: HBV-resistant mutants (rtL180M+M204V, rtV173L+L180M+M204V, rtM204I, rtL180M+M204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+N236T, and rtA181T) were produced in HepG2 cells permanently expressing the respective viral genomes. Viral protein expression, secretion, and viral particle production were studied by ELISA, Western blot, and transmission electron microscopy.

The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication.

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively.

Risk of hepatitis B surface antigen seroreversion after allogeneic hematopoietic SCT.

Allogeneic hematopoietic SCT (HSCT) increases the risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg) carriers but the incidence, risk factors and course of HBV reactivation after HSCT in HBsAg-negative/anti-hepatitis B core antigen (anti-HBc)-positive recipients are not well known. A total of 50 HBsAg-negative/anti-HBc-positive HSCT recipients with onco-hematological diseases, underwent sequential clinical and laboratory examinations, including serum HBsAg, during follow-up. Serum HBV DNA collected at HSCT was retrospectively amplified by a sensitive PCR assay.

Validation of the INNO-LiPA HBV DR assay (version 2) in monitoring hepatitis B virus-infected patients receiving nucleoside analog treatment.

Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine.

In vitro characterization of viral fitness of therapy-resistant hepatitis B variants.

Background & Aims: Because of the overlapping of polymerase and envelope genes in the hepatitis B virus (HBV) genome, nucleoside analog therapy can lead to the emergence of complex HBV variants that harbor mutations in both the reverse transcriptase and the envelope proteins. To understand the selection process of HBV variants during antiviral therapy, we analyzed the in vitro fitness (the ability to produce infectious progeny) of 4 mutant viral genomes isolated from one patient who developed resistance to a triple therapy (lamivudine, adefovir, and anti-HBV immunoglobulins).

Methods: The 4 mutant and the wild-type forms of HBV were expressed from vectors in hepatoma cell lines; replication and viral particle secretion capacities then were analyzed.

Suboptimal response to adefovir dipivoxil therapy for chronic hepatitis B in nucleoside-naive patients is not due to pre-existing drug-resistant mutants.

Background: Adefovir dipivoxil (ADV) has demonstrated activity against wild-type and lamivudine-resistant hepatitis B virus (HBV). After 1 year of therapy, a median 3.5-4.

Impact of hepatitis B virus rtA181V/T mutants on hepatitis B treatment failure.

Background/aims: Recent clinical observations reported the occurrence of amino acid substitutions at position 181 of the HBV polymerase, associated with a viral breakthrough under lamivudine or adefovir therapy. In this study, we characterized the main variants harboring the rtA181T/V mutation isolated from 10 consecutive patients who developed lamivudine and/or adefovir resistance.

Methods: We performed a clonal analysis of the HBV polymerase gene amplified by PCR from serum samples during viral breakthrough.

Characterization of a new sensitive PCR assay for quantification of viral DNA isolated from patients with hepatitis B virus infections.

Sensitive and accurate quantification of hepatitis B virus (HBV) DNA is necessary for monitoring patients with chronic hepatitis receiving antiviral therapy in order to determine treatment response and to adapt therapy in case of inadequate virologic control. The development of quantitative PCR assays has been crucial in meeting these needs. The objective of this study was to compare the performance of a new real-time PCR assay (Abbott RealTime) for HBV DNA with that of three other commercial assays for the detection of HBV DNA.

Critical role of the 36-nucleotide insertion in hepatitis B virus genotype G in core protein expression, genome replication, and virion secretion.

Frequent coinfection of hepatitis B virus genotype G with genotype A suggests that genotype G may require genotype A for replication or transmission. In this regard, genotype G is unique in having a 12-amino-acid extension in the core protein due to a 36-nucleotide insertion near the core gene translation initiation codon. The insertion alters base pairing in the lower stem of the pregenome encapsidation signal, which harbors the core gene initiator, and thus has the potential to affect both core protein translation and pregenomic RNA encapsidation.

Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient.

Background/aims: Complex mutants may be selected under sequential anti-VHB pressures. We analyzed the genotypic and phenotypic evolution of the viral quasi-species of a patient who developed resistance to entecavir following lamivudine breakthrough.

Methods: The polymerase gene was amplified, cloned and sequenced at different time points.

Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient.

Background & Aims: Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation.

Methods: For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced.

European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes.

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter.

In vitro characterization of the anti-hepatitis B virus activity and cross-resistance profile of 2',3'-dideoxy-3'-fluoroguanosine.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants.

Resistance of human hepatitis B virus to reverse transcriptase inhibitors: from genotypic to phenotypic testing.

The treatment of HBV infected patients with analogues of nucleos(t)ides, including lamivudine and adefovir dipivoxil, has significantly increased the rate of anti-HBe seroconversion and therefore reduced the impact of chronic hepatitis B (CHB) on liver disease. Altogether, these antivirals have offered novel options for the treatment of patients who did not respond to previous therapy with interferon alpha, the only available treatment against CHB until 1998. However, therapies using analogues of nucleos(t)ides have been confronted with viral resistances which are often associated to with worsening of liver disease.

Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir.

Mutations within the hepatitis B virus (HBV) polymerase gene conferring drug-resistance are selected during prolonged lamivudine (3TC) or adefovir dipivoxil (ADV) treatment. Because there is no other approved drug against HBV, treatments with 3TC or ADV are used either sequentially or in addition, depending on treatment response or failure. Considering the use of de novo or add-on 3TC+ADV bitherapy, we investigated the possibility of the emergence of an HBV strain harboring polymerase mutations conferring resistance to both 3TC (rtL180M+M204V) and ADV (rtN236T).

A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus.

Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle.

Effect of a combination of clevudine and emtricitabine with adenovirus-mediated delivery of gamma interferon in the woodchuck model of hepatitis B virus infection.

Our aim was to evaluate the antiviral effect of a combination of two nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and clevudine (L-FMAU), with the addition of an adenovirus-driven delivery of recombinant gamma interferon (IFN-gamma) in the woodchuck model of hepatitis B virus infection. Six woodchuck hepatitis virus (WHV)-infected woodchucks received L-FMAU (10 mg/kg) plus FTC (30 mg/kg) intraperitoneally for 8 weeks; six other animals received in addition an intravenous injection of a recombinant adenovirus vector expressing woodchuck IFN-gamma (Ad-IFN) at weeks 4 and 8. In the control group, two animals received Ad-IFN alone, two received adenovirus vector expressing the green fluorescent protein reporter gene, and one remained untreated.

Animal models for the study of HBV infection and the evaluation of new anti-HBV strategies.

Background: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), in study models of HBV infection.

Method: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models.

Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses.

To design combination strategies for chronic hepatitis B therapy, we evaluated in vitro the inhibitory activity of 4 nucleoside analogs, (-)FTC, L-FMAU, DXG, and DAPD, in comparison with lamivudine (3TC) and PMEA. In a cell-free assay for the expression of wild-type duck hepatitis B virus (DHBV) reverse transcriptase, DAPD-TP was found to be the most active on viral minus strand DNA synthesis, including the priming reaction, followed by 3TC-TP, (-)FTC-TP, and DXG-TP, whereas L-FMAU-TP was a weak inhibitor. In cell culture experiments, important differences in drug concentration allowing a 50% inhibition of viral replication or polymerase activity (IC50s) were observed depending on the cell type used, showing that antiviral effect of nucleoside analogs may depend on their intracellular metabolism.

Successful combination therapy of polyarteritis nodosa associated with a pre-core promoter mutant hepatitis B virus infection.

Background/aims: To describe the clinical and virological evolution of a polyarteritis nodosa (PAN) case associated with a hepatitis B virus (HBV) pre-core promoter mutant infection that was successfully treated with plasma exchanges, corticosteroids, and interferon alpha (IFN-alpha).

Methods: Viral markers were used, including HBV DNA quantified by the branched DNA assay and detected by PCR, the HBV genome sequence, pre-S1Ag and anti-HBC IgM which were studied throughout the treatment period and the entire follow-up in the serum, while the presence of virus in extrahepatic sites was detected by immuno-staining.

Results: The patient was infected with a typical pre-core promoter mutant harboring four point mutations.

Antiviral activity of beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine in woodchucks chronically infected with woodchuck hepatitis virus.

The L-nucleoside analog beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (beta-L-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob.

Duck hepatitis B virus polymerase gene mutants associated with resistance to lamivudine have a decreased replication capacity in vitro and in vivo.

Background/aims: Hepatitis B virus mutants of the polymerase gene are frequently selected during lamivudine therapy for chronic hepatitis B. To study the biology of these mutants, we analyzed their replication capacity in the duck hepatitis B virus (DHBV) infection.

Methods: The B and C domain polymerase mutants corresponding to the clinical isolates were engineered by site directed mutagenesis in the DHBV genome in different expression vectors.