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International audience; 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. Hepatoma cell lines were transfected to compare the replication capacity of HBV mutants and their drug susceptibility. RESULTS: A mixture of lamivudine-resistant HBV strains coexisted following viral breakthrough to lamivudine, all harboring the rtM204V mutation. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped but the lamivudine-resistant strains continued to be selected. Three years later, the viral load rose again, and a complex mixture of entecavir-resistant strains, all harboring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation were observed. Although the rtL180M+S202G+M204V variant, that prevailed at the end of entecavir therapy, did not show the highest viral genome replication capacity, it conferred one of the strongest resistance levels to entecavir. CONCLUSIONS: We report the selection of complex HBV mutants that escaped lamivudine and entecavir antiviral pressures. Genotypic and phenotypic analysis provided additional information to understand the process of HBV variant selection.

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.

Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-alpha) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-alpha17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-alpha2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-alpha variants or with standard IFN-alpha2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-alpha is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-alpha-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-alpha2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent "next generation" IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.

International audience; PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.