title: 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.
authors: Department of Research and Development, PhoenixBio Co., Ltd., Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors: Yuji
authors: Y
authors: Ishida
authors: Department of Research and Development, PhoenixBio Co., Ltd., Hiroshima, Japan.
authors: Chihiro
authors: C
authors: Yamasaki
authors: Department of Research and Development, PhoenixBio Co., Ltd., Hiroshima, Japan.
authors: Ami
authors: A
authors: Yanagi
authors: Department of Research and Development, PhoenixBio Co., Ltd., Hiroshima, Japan.
authors: Yasumi
authors: Y
authors: Yoshizane
authors: Division of Frontier Medical Science, Department of Medical and Molecular Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.
authors: Kazuyuki
authors: K
authors: Fujikawa
authors: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Koichi
authors: K
authors: Watashi
authors: Liver Research Project Center, Hiroshima University, Hiroshima, Japan; Division of Frontier Medical Science, Department of Medical and Molecular Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Saitama, Japan.
authors: Hiromi
authors: H
authors: Abe
authors: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Takaji
authors: T
authors: Wakita
authors: Liver Research Project Center, Hiroshima University, Hiroshima, Japan; Division of Frontier Medical Science, Department of Medical and Molecular Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Saitama, Japan.
authors: C Nelson
authors: CN
authors: Hayes
authors: Liver Research Project Center, Hiroshima University, Hiroshima, Japan; Division of Frontier Medical Science, Department of Medical and Molecular Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Saitama, Japan.
authors: Kazuaki
authors: K
authors: Chayama
authors: Department of Research and Development, PhoenixBio Co., Ltd., Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan. Electronic address: chise.mukaidani@phoenixbio.co.jp.
authors: Chise
authors: C
authors: Tateno
mesh_terms: D000818:Animals; D002675:Child, Preschool; D004279:DNA, Viral / Q000032:analysis; D004195:Disease Models, Animal; D005260:Female; D055785:Gene Knockdown Techniques; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000254:growth & development; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007223:Infant; D008297:Male; D051379:Mice; D034741:RNA, Small Interfering; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004279:DNA, Viral; D034741:RNA, Small Interfering; D012367:RNA, Viral