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  • Title: A cellular replicon-based phenotyping assay to determine susceptibility of hepatitis C virus clinical isolates to NS3/4A protease inhibitors.
    Author: Vijgen L, Verbeeck J, Van Kerckhove B, Berke JM, Koletzki D, Fanning G, Lenz O.
    Journal: Methods Mol Biol; 2013; 1030():105-17. PubMed ID: 23821263.
    Abstract:
    A hepatitis C virus (HCV) replicon-based protease phenotyping assay has been developed that allows determining the susceptibility of a patient's HCV protease sequence to HCV protease inhibitors. In brief, HCV protease sequences amplified from clinical samples are cloned in a transient HCV genotype 1b replicon backbone, containing a luciferase reporter gene. These protease chimeric replicons are replication-competent when electroporated into susceptible Huh7-Lunet cells. Replication can be quantified by measuring the enzymatic activity of the luciferase protein. This assay is reproducible and robust, and has a high overall success rate for determining the phenotypic susceptibility of HCV genotype 1a and 1b patient-derived protease domains to HCV protease inhibitors. In addition, the HCV genotype 1b protease shuttle backbone also supports efficient replication of HCV genotype 4 protease sequences.
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