Original Article Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone Norio Maeda 1 , Mamoru Watanabe 2 , Susumu Okamoto 1 , Takanori Kanai 2 , Taketo Yamada 3 , Jun-ichi Hata 3 , Nobumichi Hozumi 4 , Asao Katsume 5 , Hideko Nuriya 5 , Jasbir Sandhu 6 , Hiromasa Ishii 1 , Michinori Kohara 5 and Toshifumi Hibi 1 1 Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan, 2 Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 3 Department of Pathology, School of Medicine, Keio University, Tokyo, Japan, 4 Research Institute for Biological Sciences, Science University of Tokyo, Tokyo, Japan, 5 Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 6 Department of Surgery, Faculty of Medicine, University of Toronto, Toronto, Canada Maeda N, Watanabe M, Okamoto S, Kanai T, Yamada T, Hata J, Hozumi N, Katsume A, Nuriya H, Sandhu J, Ishii H, Kohara M, Hibi T. Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone. Liver International 2004: 24: 259–267. r Blackwell Munksgaard 2004 Abstract: Background/aims: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or ‘isolated hepatocytes’ from tree shrews. In this study, we engrafted ‘human liver tissue’ into immunodeficient mice and investigated HCV infection using an infectious molecular clone. Methods: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV- infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR. Results: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection. Conclusion: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection. Key words: hepatitis C virus – human liver tissue – infectious molecular clone – in situ PCR – NOD/SCID mouse Toshifumi Hibi, Department of Internal Medicine, Keio University School of Medicine, 35 Shina- nomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel: 81-3-3357-6286 Fax: 81-3-3357-6156 e-mail: thibi@sc.itc.keio.ac.jp Received 18 July 2003, accepted 4 February 2004                                                                                                             Hepatitis C virus (HCV) is the major causative agent of posttransfusion-associated hepatitis and of most sporadic non-A, non-B hepatitis (1). Persistent HCV infection often progresses to chronic hepatitis, liver cirrhosis and hepatocellu- lar carcinoma (2). Biological evaluation of HCV has been hampered because of the lack of small animal models for HCV infection and efficient in vitro culture systems for the virus. Recent advances in molecular cloning of HCV genome have allowed the development of infec- tious molecular clones and they have been ap- plied extensively in experimental investigation (3–6). Investigation of HCV infection using a well-characterized infectious molecular clone as the inoculum will provide valuable information to us about the underlying mechanisms of HCV infection. However, previous studies on in vivo infection of HCV molecular clones were restricted to inoculation into chimpanzees (3, 5, 6). Only a few experimental studies on HCV infection using small animal models have been reported (7, 8). Recently it was reported that HCV could infect and replicate in an immunode- ficient transgenic mouse model bearing chimeric ‘isolated human hepatocytes’ (9, 10), and this Liver International 2004: 24: 259–267 Printed in Denmark. All rights reserved Copyright r Blackwell Munksgaard 2004 DOI: 10.1111/j.1478-3231.2004.0909.x 259