Novel Robust in Vitro Hepatitis B Virus Infection Model Using Fresh Human Hepatocytes Isolated from Humanized Mice

Abstract

The molecular mechanisms underlying the hepatitis B virus (HBV) life cycle are poorly understood because of the lack of appropriate in vitro infection models. Herein, we report a highly effective in vitro HBV infection system using fresh human hepatocytes (HHs) isolated from chimeric mice with humanized livers. After the inoculation of sera collected from HBV-infected chimeric mice or patients to HHs, we measured levels of HBV DNA, mRNA, covalently closed circular DNA, and viral protein expression in HHs. We investigated the neutralization activity of hepatitis B immune globulin and the effects of siRNA against sodium taurocholate-cotransporting polypeptide and clathrin heavy chain on HBV infection. We confirmed the expression of viral antigens in HHs and the presence of extracellular HBV DNA and hepatitis B surface antigen. The maximum infection rate was approximately 80%. Lamivudine and hepatitis B immune globulin treatment reduced HBV DNA levels in a dose-dependent manner. Knockdown of sodium taurocholate-cotransporting polypeptide and clathrin heavy chain significantly reduced the levels of hepatitis B surface antigen. Infection was successfully established using different donor HHs and inocula. Elevation of extracellular HBV DNA levels and the increase of HBV-positive HHs were blocked by continuous hepatitis B immune globulin treatment, indicating virus spread in this model. Chimeric mouse-derived HHs provide a robust in vitro infection model that can completely support the HBV life cycle.

Dysregulation of Retinoic Acid Receptor Diminishes Hepatocyte Permissiveness to Hepatitis B Virus Infection through Modulation of Sodium Taurocholate Cotransporting Polypeptide (NTCP) Expression

“…It was recently reported that HBV infection in freshly isolated primary human hepatocytes could spread during long term culture through production of infectious virions and reinfection of surrounding cells (41). As shown in Fig.…”

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System

“…Huh-7, Huh7.5.1, Huh7-25, LucNeo#2, HuSE2, and 293T cells, as well as primary human hepatocytes, were cultured as described previously (2,(18)(19)(20). Huh7.5.1 and LucNeo#2 cells were kindly provided by Francis Chisari at The Scripps Research Institute and Kunitada Shimotohno at National Center for Global Health and Medicine.…”

Association between hepatitis B virus and MHC class I polypeptide-related chain A in human hepatocytes derived from human-mouse chimeric mouse liver

“…HBV genotype C isolates were purchased from PhenixBio [17,18]. Informed consent from the patient was obtained by Prof. Yasuhito Tanaka, Nagoya City University, Japan.…”

“…One day after plating, HHs were incubated with 250 mL of 2% DMSO-supplemented hepatocyte clonal growth medium (dHCGM) containing the inoculum in the presence or absence of 4% polyethylene glycol (PEG) 8000 (Promega, Tokyo, Japan) [17].…”

“…Human hepatocytes were derived from chimeric mice with hepatocyte-humanized liver (PXB-mouse), and human hepatocytes were cultured without passage as described previously [17].…”