In vitro anti-hepatitis C virus (HCV) resistance selection and characterization

Understanding the resistance profile for an antiviral drug is essential in the drug discovery process. The selection of drug-resistant mutant viruses is critical in characterizing the resistance of hepatitis C virus (HCV) to anti-HCV agents, and can be examined in HCV replicon systems. Three basic methods are employed in this unit to select HCV resistance replicons: (i) cells containing HCV replicons are cultured at low density in the presence of G418 and a fixed concentration of the investigational drug; (ii) cells containing HCV replicons are passaged in the presence of a fixed concentration of investigational drug, but in the absence of G418; (iii) cells containing HCV replicons are passaged in the presence of a gradually increasing concentration of an investigational drug in the presence of G418 for several weeks. The cells from each passage are then harvested and stored for phenotypic and genotypic characterization. The protocols in this unit describe the techniques necessary to select and characterize drug- resistant HCV variants and estimate the frequency of drug resistance in a viral population.