Hepatitis C Virus Infection of Cultured Cells and Primary Human Hepatocytes

Abstract Hepatitis C virus (HCV) is a positive‐sense, single‐stranded RNA virus in the family Flaviviridae with specific hepatotropism. HCV poses a significant health burden worldwide, frequently causing chronic infections associated with progressive liver damage and various extrahepatic manifestations. In recent years, the development of several permissive cell culture (HCVcc) systems has allowed for in vitro propagation of HCV, study of the viral life cycle and virus–host interactions, and identification of novel antivirals. Here we describe the use of human hepatoma cell lines Huh7 and HepG2/CD81/miR‐122, as well as primary human hepatocytes (PHHs), for HCV infection and propagation. We also provide protocols for the quantitative analysis of intracellular and extracellular HCV RNA and detection of HCV core protein expression by immunostaining. In addition, we describe methods to manipulate cellular gene expression, including transfection of small interfering RNAs (siRNAs) targeting HCV host factors or overexpressing cellular microRNAs in hepatocytes, to assess their effects on productive HCV infection and liver pathogenesis. © 2018 by John Wiley & Sons, Inc.