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author:("michou, C.")
1.  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. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.
doi:10.1128/AAC.00970-09
PMCID: PMC2826013  PMID: 20065049
2.  European Multicenter Evaluation of High-Density DNA Probe Arrays for Detection of Hepatitis B Virus Resistance Mutations and Identification of Genotypes 
Journal of Clinical Microbiology  2006;44(8):2792-2800.
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. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 103 genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.
doi:10.1128/JCM.00295-06
PMCID: PMC1594645  PMID: 16891494
3.  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. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.
doi:10.1128/AAC.50.3.955-961.2006
PMCID: PMC1426422  PMID: 16495257
4.  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-γ) 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-γ (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. In less than 2 weeks, all woodchucks that received L-FMAU plus FTC showed a rapid and marked inhibition of viral replication, with a 4-log10 drop in serum WHV DNA. In two animals, viremia remained suppressed for several months after the end of treatment. Similarly, a dramatic decrease in intrahepatic replicative intermediates of viral DNA was observed in the L-FMAU/FTC-treated groups. The additional administration of Ad-IFN led to increased inflammation in the liver but did not enhance the antiviral effect of the L-FMAU/FTC combination. In conclusion, therapies combining L-FMAU and FTC in WHV-infected woodchucks resulted in a potent and sustained antihepadnaviral effect both in the liver and in the blood circulation. However, no extra benefit of adding IFN-γ gene transduction to the L-FMAU/FTC combination could be detected.
doi:10.1128/AAC.48.7.2683-2692.2004
PMCID: PMC434178  PMID: 15215126
5.  Antiviral Activity of β-l-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus 
The l-nucleoside analog β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-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. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-l-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-l-Fd4C-treated groups. Moreover, β-l-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-l-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-l-Fd4C treatment. There was no evidence that lamivudine or β-l-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-l-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
doi:10.1128/AAC.45.4.1065-1077.2001
PMCID: PMC90426  PMID: 11257017
6.  Diagnostic markers of viral hepatitis B and C. 
Gut  1993;34(2 Suppl):S20-S25.
Hepatitis B virus (HBV) serology has become extremely refined. As well as the recognised hepatitis B surface (HBs), hepatitis B core (HBc), and hepatitis B e (HBe) antigen-antibody systems, new markers have been introduced including pre-S1, pre-S2 for the envelope and the functional X protein. New automates have been introduced allowing flexibility in the different tests according to precise needs. The monitoring of pre-S1 antigen provides a relevant correlate of viral replication. The quantitative determination of HBV-DNA, pre-S1 Ag, and IgM anti-HBc seem most useful for the decision to use, and the monitoring of, antiviral treatment. Second generation ELISAs detect antibodies to three sets of hepatitis C virus (HCV) protein including the c22 core, and c33, and c100, which correspond to the non-structural regions (NS3 and NS4, respectively). Second generation ELISAs require confirmation by supplement assays, but their biggest limitation is the delayed appearance of anti-HCV after primary infection. In addition 10% of chronic infections with liver disease still remain seronegative despite circulating HCV RNA in serum or liver, or both. Much progress still has to be made before HCV serology can reach the level of sophistication of HBV.
PMCID: PMC1373999  PMID: 8314489
7.  New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection. 
Journal of Clinical Microbiology  1992;30(5):1111-1119.
We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.
PMCID: PMC265234  PMID: 1583107
8.  Comparison of properties of woodchuck hepatitis virus and human hepatitis B virus endogenous DNA polymerases. 
The principal properties of the DNA polymerases of woodchuck hepatitis virus and human hepatitis B virus were compared. The enzymes of both viruses exhibited optimal activities in the same range of pH, ionic strength, and MgCl2 concentration. Like human hepatitis B virus DNA polymerase, the woodchuck hepatitis virus DNA polymerase was strongly inhibited by phosphonoformic acid but not by phosphonoacetic acid and aphidicolin. Similar inhibition patterns for both enzymes were observed with arabinofuranosyl nucleotides (9-beta-D-arabinofuranosyladenine-5'-triphosphate, 1-beta-D-arabinofuranosylcytosine-5'-triphosphate, 1-beta-D-arabinofuranosylthymine-5'-triphosphate) and dideoxythymidine triphosphate, whereas no effect was obtained with corresponding nucleosides. The therapeutic significance of these results and the relevance of the woodchuck as an experimental animal model for the study of human hepatitis B virus infections are discussed.
PMCID: PMC185482  PMID: 6231885

Results 1-8 (8)