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Song J, Sun X, Zhou Y, Li S, Wu J, Yang L, Zhou D, Yang Y, Liu A, Lu M, Michael R, Qin L, Yang D. Early application of IFNγ mediated the persistence of HBV in an HBV mouse model. Antiviral Res 2024; 225:105872. [PMID: 38556058 DOI: 10.1016/j.antiviral.2024.105872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Revised: 03/20/2024] [Accepted: 03/23/2024] [Indexed: 04/02/2024]
Abstract
The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.
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Affiliation(s)
- Jingjiao Song
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Xiliang Sun
- Clinical Laboratory, Qingdao West Coast New District People's Hospital, Shandong, PR China.
| | - Yun Zhou
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Key Laboratory of Receptors-mediated Gene Regulation and Drug Discovery, School of Medicine, Henan University, Kaifeng, PR China.
| | - Sheng Li
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Jun Wu
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Lu Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Di Zhou
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Yan Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Anding Liu
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.
| | | | - Li Qin
- Department of Dermatology, Traditional Chinese and Western Medicine Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei, PR China.
| | - Dongliang Yang
- Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
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Protocol for chronic hepatitis B virus infection mouse model development by patient-derived orthotopic xenografts. PLoS One 2022; 17:e0264266. [PMID: 35196351 PMCID: PMC8865695 DOI: 10.1371/journal.pone.0264266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Accepted: 02/01/2022] [Indexed: 12/03/2022] Open
Abstract
Background According to the World Health Organization, more than 250 million people worldwide are chronically infected with the hepatitis B virus, and almost 800.000 patients die annually of mediated liver disorders. Therefore, adequate biological test systems are needed that could fully simulate the course of chronic hepatitis B virus infection, including in patients with hepatocellular carcinoma. Methods In this study, we will assess the effectiveness of existing protocols for isolation and cultivation of primary cells derived from patients with hepatocellular carcinoma in terms of the yield of viable cells and their ability to replicate the hepatitis B virus using isolation and cultivation methods for adhesive primary cells, flow cytometry and quantitative polymerase chain reaction. Another part of our study will be devoted to evaluating the effectiveness of hepatocellular carcinoma grafting methods to obtain patient-derived heterotopic and orthotopic xenograft mouse avatars using animal X-ray irradiation and surgery procedures and in vivo fluorescent signals visualization and measurements. Our study will be completed by histological methods. Discussion This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice. The most effective methods will be recommended for use in translational biomedical research.
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Avdoshina DV, Kondrashova AS, Belikova MG, Bayurova EO. Murine Models of Chronic Viral Infections and Associated Cancers. Mol Biol 2022; 56:649-667. [PMID: 36217336 PMCID: PMC9534466 DOI: 10.1134/s0026893322050028] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 04/12/2022] [Accepted: 04/13/2022] [Indexed: 11/07/2022]
Abstract
Viruses are now recognized as bona fide etiologic factors of human cancer. Carcinogenic viruses include Epstein– Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus type 1 (HIV-1, indirectly), and several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. Tumor viruses establish long-term persistent infections in humans, and cancer is an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, supporting the concept that cancer results from the accumulation of multiple cooperating events, in which human cancer viruses display different, often opposing roles. The laboratory mouse Mus musculus is one of the best in vivo experimental systems for modeling human pathology, including viral infections and cancer. However, mice are unsusceptible to infection with the known carcinogenic viruses. Many murine models were developed to overcome this limitation and to address various aspects of virus-associated carcinogenesis, from tumors resulting from xenografts of human tissues and cells, including cancerous and virus infected, to genetically engineered mice susceptible to viral infections and associated cancer. The review considers the main existing models, analyzes their advantages and drawbacks, describes their applications, outlines the prospects of their further development.
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Affiliation(s)
- D. V. Avdoshina
- Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), 108819 Moscow, Russia
| | - A. S. Kondrashova
- Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), 108819 Moscow, Russia
| | - M. G. Belikova
- Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), 108819 Moscow, Russia ,Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia ,Peoples’ Friendship University of Russia, 117198 Moscow, Russia
| | - E. O. Bayurova
- Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), 108819 Moscow, Russia ,Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia
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4
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Zhang X, Wang X, Wu M, Ghildyal R, Yuan Z. Animal Models for the Study of Hepatitis B Virus Pathobiology and Immunity: Past, Present, and Future. Front Microbiol 2021; 12:715450. [PMID: 34335553 PMCID: PMC8322840 DOI: 10.3389/fmicb.2021.715450] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Accepted: 06/18/2021] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) infection is a global public health problem that plagues approximately 240 million people. Chronic hepatitis B (CHB) often leads to liver inflammation and aberrant repair which results in diseases ranging from liver fibrosis, cirrhosis, to hepatocellular carcinoma. Despite its narrow species tropism, researchers have established various in vivo models for HBV or its related viruses which have provided a wealth of knowledge on viral lifecycle, pathogenesis, and immunity. Here we briefly revisit over five decades of endeavor in animal model development for HBV and summarize their advantages and limitations. We also suggest directions for further improvements that are crucial for elucidation of the viral immune-evasion strategies and for development of novel therapeutics for a functional cure.
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Affiliation(s)
- Xiaonan Zhang
- Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Canberra, ACT, Australia.,Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Xiaomeng Wang
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Min Wu
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Reena Ghildyal
- Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Canberra, ACT, Australia
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
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5
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Rao S, Hossain T, Mahmoudi T. 3D human liver organoids: An in vitro platform to investigate HBV infection, replication and liver tumorigenesis. Cancer Lett 2021; 506:35-44. [PMID: 33675983 DOI: 10.1016/j.canlet.2021.02.024] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 02/18/2021] [Accepted: 02/25/2021] [Indexed: 02/07/2023]
Abstract
Hepatitis B Virus (HBV) infection is a leading cause of chronic liver cirrhosis and hepatocellular carcinoma (HCC) with an estimated 400 million people infected worldwide. The precise molecular mechanisms underlying HBV replication and tumorigenesis have remained largely uncharacterized due to the lack of a primary cell model to study HBV, a virus that exhibits stringent host species and cell-type specificity. Organoid technology has recently emerged as a powerful tool to investigate human diseases in a primary 3D cell-culture system that maintains the organisation and functionality of the tissue of origin. In this review, we describe the utilisation of human liver organoid platforms to study HBV. We first present the different categories of liver organoids and their demonstrated ability to support the complete HBV replication cycle. We then discuss the potential applications of liver organoids in investigating HBV infection and replication, related tumorigenesis and novel HBV-directed therapies. Liver organoids can be genetically modified, patient-derived, expanded and biobanked, thereby serving as a clinically-relevant, human, primary cell-derived platform to investigate HBV. Finally, we provide insights into the future applications of this powerful technology in the context of HBV-infection and HCC.
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Affiliation(s)
- Shringar Rao
- Department of Biochemistry, Erasmus University Medical Centre, PO Box 2040, 3000, CA, 9 Rotterdam, the Netherlands
| | - Tanvir Hossain
- Department of Biochemistry, Erasmus University Medical Centre, PO Box 2040, 3000, CA, 9 Rotterdam, the Netherlands
| | - Tokameh Mahmoudi
- Department of Biochemistry, Erasmus University Medical Centre, PO Box 2040, 3000, CA, 9 Rotterdam, the Netherlands; Department of Pathology, Erasmus University Medical Centre, PO Box 2040, 3000, CA, Rotterdam, the Netherlands; Department of Urology, Erasmus University Medical Centre, PO Box 2040, 3000, CA, Rotterdam, the Netherlands.
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6
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Lai F, Wee CYY, Chen Q. Establishment of Humanized Mice for the Study of HBV. Front Immunol 2021; 12:638447. [PMID: 33679796 PMCID: PMC7933441 DOI: 10.3389/fimmu.2021.638447] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2020] [Accepted: 02/03/2021] [Indexed: 12/28/2022] Open
Abstract
Viral hepatitis particularly Hepatitis B Virus (HBV) is still an ongoing health issue worldwide. Despite the vast technological advancements in research and development, only HBV vaccines, typically given during early years, are currently available as a preventive measure against acquiring the disease from a secondary source. In general, HBV can be cleared naturally by the human immune system if detected at low levels early. However, long term circulation of HBV in the peripheral blood may be detrimental to the human liver, specifically targeting human hepatocytes for cccDNA integration which inevitably supports HBV life cycle for the purpose of reinfection in healthy cells. Although there is some success in using nucleoside analogs or polyclonal antibodies targeting HBV surface antigens (HBsAg) in patients with acute or chronic HBV+ (CHB), majority of them would either respond only partially or succumb to the disease entirely unless they undergo liver transplants from a fully matched healthy donor and even so may not necessarily guarantee a 100% chance of survival. Indeed, in vitro/ex vivo cultures and various transgenic animal models have already provided us with a good understanding of HBV but they primarily lack human specificity or virus-host interactions in the presence of human immune surveillance. Therefore, the demand of utilizing humanized mice has increased over the last decade as a pre-clinical platform for investigating human-specific immune responses against HBV as well as identifying potential immunotherapeutic strategies in eradicating the virus. Basically, this review covers some of the recent developments and key advantages of humanized mouse models over other conventional transgenic mice platforms.
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Affiliation(s)
- Fritz Lai
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Cherry Yong Yi Wee
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
| | - Qingfeng Chen
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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7
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Hayes CN, Chayama K. Unmet Needs in Basic Research of Hepatitis B Virus Infection: In Vitro and In Vivo Models. HEPATITIS B VIRUS AND LIVER DISEASE 2021:29-49. [DOI: 10.1007/978-981-16-3615-8_2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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Ely A, Singh P, Smith TS, Arbuthnot P. In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection. Adv Drug Deliv Rev 2021; 168:134-146. [PMID: 32485207 DOI: 10.1016/j.addr.2020.05.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Revised: 05/14/2020] [Accepted: 05/27/2020] [Indexed: 02/06/2023]
Abstract
Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.
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9
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Mouse models for hepatitis B virus research. Lab Anim Res 2018; 34:85-91. [PMID: 30310404 PMCID: PMC6170223 DOI: 10.5625/lar.2018.34.3.85] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2018] [Revised: 09/13/2018] [Accepted: 09/13/2018] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) infection remains a major global health problem; indeed, there are 250 million carriers worldwide. The host range of HBV is narrow; therefore, few primates are susceptible to HBV infection. However, ethical constraints, high cost, and large size limit the use of primates as suitable animal models. Thus, in vivo testing of therapies that target HBV has been hampered by the lack of an appropriate in vivo research model. To address this, mouse model systems of HBV are being developed and several are used for studying HBV in vivo. In this review, we summarize the currently available mouse models, including HBV transgenic mice, hydrodynamic injection-mediated HBV replicon delivery systems, adeno-associated virus-mediated HBV replicon delivery systems, and human liver chimeric mouse models. These developed (or being developed) mouse model systems are promising and should be useful tools for studying HBV.
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Burm R, Collignon L, Mesalam AA, Meuleman P. Animal Models to Study Hepatitis C Virus Infection. Front Immunol 2018; 9:1032. [PMID: 29867998 PMCID: PMC5960670 DOI: 10.3389/fimmu.2018.01032] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Accepted: 04/25/2018] [Indexed: 12/18/2022] Open
Abstract
With more than 71 million chronically infected people, the hepatitis C virus (HCV) is a major global health concern. Although new direct acting antivirals have significantly improved the rate of HCV cure, high therapy cost, potential emergence of drug-resistant viral variants, and unavailability of a protective vaccine represent challenges for complete HCV eradication. Relevant animal models are required, and additional development remains necessary, to effectively study HCV biology, virus–host interactions and for the evaluation of new antiviral approaches and prophylactic vaccines. The chimpanzee, the only non-human primate susceptible to experimental HCV infection, has been used extensively to study HCV infection, particularly to analyze the innate and adaptive immune response upon infection. However, financial, practical, and especially ethical constraints have urged the exploration of alternative small animal models. These include different types of transgenic mice, immunodeficient mice of which the liver is engrafted with human hepatocytes (humanized mice) and, more recently, immunocompetent rodents that are susceptible to infection with viruses that are closely related to HCV. In this review, we provide an overview of the currently available animal models that have proven valuable for the study of HCV, and discuss their main benefits and weaknesses.
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Affiliation(s)
- Rani Burm
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Laura Collignon
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Ahmed Atef Mesalam
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium.,Therapeutic Chemistry Department, National Research Centre (NRC), Cairo, Egypt
| | - Philip Meuleman
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
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Chayama K, Nelson Hayes C. Unmet Needs in Basic Research: In Vitro and In Vivo Models. HEPATITIS B VIRUS AND LIVER DISEASE 2018:25-43. [DOI: 10.1007/978-981-10-4843-2_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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12
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Gural N, Mancio-Silva L, He J, Bhatia SN. Engineered Livers for Infectious Diseases. Cell Mol Gastroenterol Hepatol 2017; 5:131-144. [PMID: 29322086 PMCID: PMC5756057 DOI: 10.1016/j.jcmgh.2017.11.005] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2017] [Accepted: 11/02/2017] [Indexed: 01/18/2023]
Abstract
Engineered liver systems come in a variety of platform models, from 2-dimensional cocultures of primary human hepatocytes and stem cell-derived progeny, to 3-dimensional organoids and humanized mice. Because of the species-specificity of many human hepatropic pathogens, these engineered systems have been essential tools for biologic discovery and therapeutic agent development in the context of liver-dependent infectious diseases. Although improvement of existing models is always beneficial, and the addition of a robust immune component is a particular need, at present, considerable progress has been made using this combination of research platforms. We highlight advances in the study of hepatitis B and C viruses and malaria-causing Plasmodium falciparum and Plasmodium vivax parasites, and underscore the importance of pairing the most appropriate model system and readout modality with the particular experimental question at hand, without always requiring a platform that recapitulates human physiology in its entirety.
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Key Words
- 2D, 2-dimensional
- 3D
- 3D, 3-dimensional
- EBOV, Ebola virus
- Falciparum
- HBC, hepatitis C virus
- HBV
- HBV, hepatitis B virus
- HCV
- HLC, hepatocyte-like cells
- Hepatotropic
- LASV, Lassa virus
- Liver
- Liver Models
- MPCC, micropatterned coculture system
- Malaria
- PCR, polymerase chain reaction
- Pathogen
- SACC, self-assembling coculture
- Vivax
- iHLC, induced pluripotent stem cell–derived hepatocyte-like cells
- in vitro
- in vivo
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Affiliation(s)
- Nil Gural
- Harvard-MIT Department of Health Sciences and Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Boston, Massachusetts,Koch Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Liliana Mancio-Silva
- Koch Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts,Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Jiang He
- Koch Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts,Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Sangeeta N. Bhatia
- Koch Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts,Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts,Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts,Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts,Broad Institute, Cambridge, Massachusetts,Howard Hughes Medical Institute, Chevy Chase, Maryland,Correspondence Address correspondence to: Sangeeta N. Bhatia, MD, PhD, Koch Institute for Integrative Cancer, Research at MIT, Building 76, Room 473, 500 Main Street, Cambridge, Massachusetts 02142.
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Li L, Li S, Zhou Y, Yang L, Zhou D, Yang Y, Lu M, Yang D, Song J. The dose of HBV genome contained plasmid has a great impact on HBV persistence in hydrodynamic injection mouse model. Virol J 2017; 14:205. [PMID: 29070073 PMCID: PMC5657044 DOI: 10.1186/s12985-017-0874-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2017] [Accepted: 10/18/2017] [Indexed: 01/12/2023] Open
Abstract
Background Hydrodynamic injection (HI) of hepatitis B virus (HBV) mouse model is an useful tool for HBV related research in vivo. However, only 40% of C57/BL6 mice injected with 10 μg HBV genome contained plasmid (pAAV-HBV1.2), serum HBsAg more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks in the previous study. Methods In this study, C57/BL6 and BALB/c mice were hydrodynamic injected with different doses of pAAV-HBV1.2 plasmid DNA. HBV related serum markers were detected by ELISA. ALT levels in the serum were measured using full automated biochemistry analyzer. HBcAg positive cells in the liver were detected by immunohistochemical staining. The mRNA levels of IRF3, ISGs including ISG15, OAS, PKR and immune factors including IFNγ, TNFα, TGFβ, IL-6, IL-10, PDL1 in liver of the mice were quantified by qRT-PCR. Results The results showed that the mice injected with 100 μg high-concentration or 1 μg low-concentration of pAAV-HBV1.2 plasmid DNA did not excert dominant influence on HBV persistence. In contrast, injection of 5 μg intermediate-dose of pAAV-HBV1.2 plasmid DNA led to significant prolonged HBsAg expression and HBV persistence in both C57/BL6 (80% of the mice with HBsAg positive more than 6 months) and BALB/c (60% of the mice with HBsAg positive more than 3 months) mice. IFNγ was significant up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Moreover, PDL1 was significant up-regulated in liver of the mice injected with 5 μg pAAV-HBV1.2 plasmid DNA. Conclusion In this paper we demonstrated that, in the HBV HI mouse model, the concentration of injected pAAV-HBV1.2 plasmid DNA contributes to the diverse kinetics of HBsAg and HBeAg in the serum as well as HBcAg expression level in the liver, which then determined the HBV persisternce, while the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence.
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Affiliation(s)
- Lei Li
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.,Department of Infectious Disease, Anhui Provincial Hospital, Anhui Medical University, Hefei, China
| | - Sheng Li
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.,Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Yun Zhou
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.,Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lu Yang
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Di Zhou
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Yan Yang
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
| | - Dongliang Yang
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jingjiao Song
- Experimental Medicine Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
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14
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Sun S, Li J. Humanized chimeric mouse models of hepatitis B virus infection. Int J Infect Dis 2017; 59:131-136. [PMID: 28408253 DOI: 10.1016/j.ijid.2017.04.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2017] [Revised: 04/01/2017] [Accepted: 04/03/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) infection is associated with an increased risk of hepatic cirrhosis, hepatocellular carcinoma, fulminant hepatitis and end-stage hepatic failure. Despite the availability of anti-HBV therapies, HBV infection remains a major global public health problem. Developing an ideal animal model of HBV infection to clarify the details of the HBV replication process, the viral life cycle, the resulting immunoresponse and the precise pathogenesis of HBV is difficult because HBV has an extremely narrow host range and almost exclusively infects humans. In this review, we summarize and evaluate animal models available for studying HBV infection, especially focusing on humanized chimeric mouse models, and we discuss future development trends regarding immunocompetent humanized mouse models that can delineate the natural history and immunopathophysiology of HBV infection.
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Affiliation(s)
- Suwan Sun
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Rd., Hangzhou 310003, China
| | - Jun Li
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Rd., Hangzhou 310003, China.
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15
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A New Oleanolic Acid Derivative against CCl₄-Induced Hepatic Fibrosis in Rats. Int J Mol Sci 2017; 18:ijms18030553. [PMID: 28272302 PMCID: PMC5372569 DOI: 10.3390/ijms18030553] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2017] [Revised: 02/21/2017] [Accepted: 02/26/2017] [Indexed: 12/12/2022] Open
Abstract
A novel hepatoprotective oleanolic acid derivative, 3-oxours-oleana-9(11), 12-dien-28-oic acid (Oxy-Di-OA), has been reported. In previous studies, we found that Oxy-Di-OA presented the anti-HBV (Hepatitis B Virus) activity (IC50 = 3.13 µg/mL). Remarkably, it is superior to lamivudine in the inhibition of the rebound of the viral replication rate. Furthermore, Oxy-Di-OA showed good performance of anti-HBV activity in vivo. Some studies showed that liver fibrosis may affiliate with HBV gene mutations. In addition, the anti-hepatic fibrosis activity of Oxy-Di-OA has not been studied. Therefore, we evaluated the protective effect of Oxy-Di-OA against carbon tetrachloride (CCl4)-induced liver injury in rats. Daily intraperitoneally administration of Oxy-Di-OA prevented the development of CCl4-induced liver fibrosis, which was evidenced by histological study and immunohistochemical analysis. The entire experimental protocol lasted nine weeks. Oxy-Di-OA significantly suppressed the increases of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (p < 0.05). Furthermore, Oxy-Di-OA could prevent expression of transforming growth factor β1 (TGF-β1). It is worth noting that the high-dose group Oxy-Di-OA is superior to bifendate in elevating hepatic function. Compared to the model group, Oxy-Di-OA in the high-dose group and low-dose group can significantly reduce the liver and spleen indices (p < 0.05). The acute toxicity test showed that LD50 and a 95% confidence interval (CIs) value of Oxy-Di-OA were 714.83 mg/kg and 639.73–798.73 mg/kg via intraperitoneal injection in mice, respectively. The LD50 value of Oxy-Di-OA exceeded 2000 mg/kg via gavage in mice. In addition, a simple and rapid high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated to study the pharmacokinetic characteristics of the compound. After single-dose oral administration, time to reach peak concentration of Oxy-Di-OA (Cmax = 8.18 ± 0.66 μg/mL) was 10 ± 2.19 h; the elimination half-life and area under the concentration-time curve from t = 0 to the last time of Oxy-Di-OA was 2.19 h and 90.21 μg·h/mL, respectively.
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16
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Teng YC, Shen ZQ, Kao CH, Tsai TF. Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes. World J Gastroenterol 2016; 22:300-325. [PMID: 26755878 PMCID: PMC4698494 DOI: 10.3748/wjg.v22.i1.300] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Revised: 10/14/2015] [Accepted: 11/24/2015] [Indexed: 02/06/2023] Open
Abstract
The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine.
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17
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Iannacone M, Guidotti LG. Mouse Models of Hepatitis B Virus Pathogenesis. Cold Spring Harb Perspect Med 2015; 5:cshperspect.a021477. [PMID: 26292984 DOI: 10.1101/cshperspect.a021477] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The host range of hepatitis B virus (HBV) is limited to humans and chimpanzees. As discussed in the literature, numerous studies in humans and chimpanzees have generated a great deal of information on the mechanisms that cause viral clearance, viral persistence, and disease pathogenesis during acute or chronic HBV infection. Relevant pathogenetic studies have also been performed in those few species representing natural hosts of hepadnaviruses that are related to HBV, such as the woodchuck hepatitis virus and the duck hepatitis virus. Further insight has been gained from multidisciplinary studies in transgenic or humanized chimeric mouse models expressing and/or replicating HBV to varying degrees. We provide here a concise summary of the available HBV mouse models as well as of the contributions of these models to our understanding of HBV pathogenesis.
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Affiliation(s)
- Matteo Iannacone
- Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy Vita-Salute San Raffaele University, 20132 Milan, Italy
| | - Luca G Guidotti
- Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy Department of Immunology & Microbial Sciences, The Scripps Research Institute, La Jolla, California 92037
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18
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Cheng L, Li F, Bility MT, Murphy CM, Su L. Modeling hepatitis B virus infection, immunopathology and therapy in mice. Antiviral Res 2015; 121:1-8. [PMID: 26099683 DOI: 10.1016/j.antiviral.2015.06.012] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2015] [Revised: 06/16/2015] [Accepted: 06/18/2015] [Indexed: 12/19/2022]
Abstract
Despite the availability of a preventive vaccine, chronic hepatitis B virus (HBV) infection-induced liver diseases continue to be a major global public health problem. HBV naturally infects only humans and chimpanzees. This narrow host range has hindered our ability to study the characteristics of the virus and how it interacts with its host. It is thus important to establish small animal models to study HBV infection, persistence, clearance and the immunopathogenesis of chronic hepatitis B. In this review, we briefly summarize currently available animal models for HBV research, then focus on mouse models, especially the recently developed humanized mice that can support HBV infection and immunopathogenesis in vivo. This article is part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story."
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Affiliation(s)
- Liang Cheng
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina, Chapel Hill, NC, USA
| | - Feng Li
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina, Chapel Hill, NC, USA
| | - Moses T Bility
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina, Chapel Hill, NC, USA
| | - Christopher M Murphy
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina, Chapel Hill, NC, USA
| | - Lishan Su
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina, Chapel Hill, NC, USA.
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19
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Dembek C, Protzer U. Mouse models for therapeutic vaccination against hepatitis B virus. Med Microbiol Immunol 2015; 204:95-102. [PMID: 25523197 DOI: 10.1007/s00430-014-0378-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Accepted: 10/02/2014] [Indexed: 02/07/2023]
Abstract
A mouse model for persistent HBV infection is essential for the development of a therapeutic vaccine against HBV. Because HBV cannot infect mouse hepatocytes, even if the HBV receptor is introduced, surrogate models are used. A suitable model needs to establish persistent HBV replication and must allow the establishment of HBV-specific adaptive cellular and humoral immune responses. Therefore, an immunocompetent mouse model is needed in which one can break HBV-specific tolerance and ideally eliminate the HBV transcription template. The most widely used model for chronic HBV infection is the HBV transgenic mouse. Although HBV replicates from an integrated transgene, HBV-specific immune tolerance can be broken upon adequate immune stimulation because antigen expression only starts shortly before birth. Alternative mouse models of chronic HBV infection are generated by introducing HBV genomes either using viral vectors or using hydrodynamic injection. In these alternative models, the HBV transcription template is introduced into a proportion of hepatocytes and stays extra-chromosomal. It thus mimics the natural HBV transcription template, the HBV cccDNA in humans. Unlike an HBV transgene, however, it can be cleared upon appropriate treatment or immune stimulation. Human hepatocyte chimeric mice in which murine hepatocytes are widely replaced by human hepatocytes represent another important mouse model for persistent HBV infection. These mice are susceptible for HBV infection, but need to be severely immune deficient to accept human hepatocytes. In conclusion, a variety of mouse models for persistent HBV infection are available suitable for preclinical efficacy evaluations of therapeutic vaccination strategies against HBV.
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Affiliation(s)
- Claudia Dembek
- Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675, Munich, Germany
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20
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Inuzuka T, Takahashi K, Chiba T, Marusawa H. Mouse models of hepatitis B virus infection comprising host-virus immunologic interactions. Pathogens 2014; 3:377-89. [PMID: 25437805 PMCID: PMC4243451 DOI: 10.3390/pathogens3020377] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Revised: 04/09/2014] [Accepted: 04/11/2014] [Indexed: 12/13/2022] Open
Abstract
Hepatitis B virus (HBV) infection is one of the most prevalent infectious diseases associated with various human liver diseases, including acute, fulminant and chronic hepatitis; liver cirrhosis; and hepatocellular carcinoma. Despite the availability of an HBV vaccine and the development of antiviral therapies, there are still more than 350 million chronically infected people worldwide, approximately 5% of the world population. To understand the virus biology and pathogenesis in HBV-infected patients, several animal models have been developed to mimic hepatic HBV infection and the immune response against HBV, but the narrow host range of HBV infection and lack of a full immune response spectrum in animal models remain significant limitations. Accumulating evidence obtained from studies using a variety of mouse models that recapitulate hepatic HBV infection provides several clues for understanding host-virus immunologic interactions during HBV infection, whereas the determinants of the immune response required for HBV clearance are poorly defined. Therefore, adequate mouse models are urgently needed to elucidate the mechanism of HBV elimination and identify novel targets for antiviral therapies.
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Affiliation(s)
- Tadashi Inuzuka
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8103, Japan.
| | - Ken Takahashi
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8103, Japan.
| | - Tsutomu Chiba
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8103, Japan.
| | - Hiroyuki Marusawa
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8103, Japan.
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Dandri M, Lütgehetmann M. Mouse models of hepatitis B and delta virus infection. J Immunol Methods 2014; 410:39-49. [PMID: 24631647 DOI: 10.1016/j.jim.2014.03.002] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2014] [Revised: 03/03/2014] [Accepted: 03/04/2014] [Indexed: 01/05/2023]
Abstract
Liver disease associated to persistent infection with the hepatitis B virus (HBV) continues to be a major health problem of global impact. Therapeutic regimens currently available can efficiently suppress HBV replication; however, the unique replication strategies employed by HBV permit the virus to persist within the infected hepatocytes. As a consequence, relapse of viral activity is commonly observed after cessation of treatment with polymerase inhibitors. Among the HBV chronically infected patients, more than 15million patients are estimated to be co-infected with the hepatitis delta virus (HDV), a defective satellite virus that needs the HBV envelope for propagation. No specific drugs are currently available against HDV, while nucleos(t)ide analogs are not effective against HDV replication. Since chronic HBV/HDV co-infection leads to the most severe form of chronic viral hepatitis in men, a better understanding of the molecular mechanisms of HDV-mediated pathogenesis and the development of improved therapeutic approaches is urgently needed. The obvious limitations imposed by the use of great apes and the paucity of robust experimental models of HBV infection have hindered progresses in understanding the complex network of virus-host interactions that are established in the course of HBV and HDV infections. This review focuses on summarizing recent advances obtained with well-established and more innovative experimental mouse models, giving emphasis on the strength of infection systems based on the reconstitution of the murine liver with human hepatocytes, as tools for elucidating the whole life cycle of HBV and HDV, as well as for studies on interactions with the infected human hepatocytes and for preclinical drug evaluation.
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Affiliation(s)
- Maura Dandri
- I. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; German Center for Infection Research, Hamburg-Lübeck-Borstel Partner Site, Germany.
| | - Marc Lütgehetmann
- I. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Institute of Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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22
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Li L, Shen H, Li A, Zhang Z, Wang B, Wang J, Zheng X, Wu J, Yang D, Lu M, Song J. Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B. Virol J 2013; 10:214. [PMID: 23805945 PMCID: PMC3751867 DOI: 10.1186/1743-422x-10-214] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Accepted: 06/21/2013] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND It has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI. METHODS We constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model. RESULTS The 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective. CONCLUSIONS Taken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo.
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Affiliation(s)
- Lei Li
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
- Department of Infectious Disease, Anhui Provincial Hospital, No.9 Lujiang Road, Hefei, P.R. China
| | - Hong Shen
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Anyi Li
- Animal Center, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Zhenhua Zhang
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Baoju Wang
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Junzhong Wang
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Xin Zheng
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Jun Wu
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Dongliang Yang
- Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
| | - Jingjiao Song
- Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China
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23
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Liu Y, Meyer C, Xu C, Weng H, Hellerbrand C, ten Dijke P, Dooley S. Animal models of chronic liver diseases. Am J Physiol Gastrointest Liver Physiol 2013; 304:G449-G468. [PMID: 23275613 DOI: 10.1152/ajpgi.00199.2012] [Citation(s) in RCA: 150] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Chronic liver diseases are frequent and potentially life threatening for humans. The underlying etiologies are diverse, ranging from viral infections, autoimmune disorders, and intoxications (including alcohol abuse) to imbalanced diets. Although at early stages of disease the liver regenerates in the absence of the insult, advanced stages cannot be healed and may require organ transplantation. A better understanding of underlying mechanisms is mandatory for the design of new drugs to be used in clinic. Therefore, rodent models are being developed to mimic human liver disease. However, no model to date can completely recapitulate the "corresponding" human disorder. Limiting factors are the time frame required in humans to establish a certain liver disease and the fact that rodents possess a distinct immune system compared with humans and have different metabolic rates affecting liver homeostasis. These features account for the difficulties in developing adequate rodent models for studying disease progression and for testing new pharmaceuticals to be translated into the clinic. Nevertheless, traditional and new promising animal models that mimic certain attributes of chronic liver diseases are established and being used to deepen our understanding in the underlying mechanisms of distinct liver diseases. This review aims at providing a comprehensive overview of recent advances in animal models recapitulating different features and etiologies of human liver diseases.
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Affiliation(s)
- Yan Liu
- Department of Medicine II, Section Molecular Hepatology-Alcohol Associated Diseases, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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24
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Abstract
Hepatitis B virus (HBV), a small and economically packaged double-stranded DNA virus, represents an enormous global health care burden. In spite of an effective vaccine, HBV is endemic in many countries. Chronic hepatitis B (CHB) results in the development of significant clinical outcomes such as liver disease and hepatocellular carcinoma (HCC), which are associated with high mortality rates. HBV is a non-cytopathic virus, with the host's immune response responsible for the associated liver damage. Indeed, HBV appears to be a master of manipulating and modulating the immune response to achieve persistent and chronic infection. The HBV precore protein or hepatitis B e antigen (HBeAg) is a key viral protein involved in these processes, for instance though the down-regulation of the innate immune response. The development of new therapies that target viral proteins, such as HBeAg, which regulates of the immune system, may offer a new wave of potential therapeutics to circumvent progression to CHB and liver disease.
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Affiliation(s)
- Renae Walsh
- Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia.
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25
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Dandri M, Lütgehetmann M, Petersen J. Experimental models and therapeutic approaches for HBV. Semin Immunopathol 2012; 35:7-21. [PMID: 22898798 DOI: 10.1007/s00281-012-0335-7] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2012] [Accepted: 07/31/2012] [Indexed: 12/12/2022]
Abstract
Liver disease associated to persistent infection with the hepatitis B virus (HBV) continues to be a major health problem of global impact. In spite of the existence of an effective vaccine, approximately 360 million people are chronically infected worldwide, who are at high risk of developing liver cirrhosis and hepatocellular carcinoma. Although current therapeutic regimens can efficiently suppress viral replication, the unique replication strategies employed by HBV permit the virus to persist within the infected hepatocytes. As a consequence, relapse of viral activity is commonly observed after cessation of treatment with polymerase inhibitors. The narrow host range of HBV has hindered progresses in understanding specific steps of HBV replication and the development of more effective therapeutic strategies aiming at achieving sustained viral control and, eventually, virus eradication. This review will focus on summarizing recent advances obtained with well-established and more innovative experimental models, giving emphasis on the strength of the different systems as tools for elucidating distinct aspects of HBV persistence and for the development of new therapeutic approaches.
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Affiliation(s)
- Maura Dandri
- Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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26
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Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis. J Virol 2012; 86:9443-53. [PMID: 22740403 DOI: 10.1128/jvi.00873-12] [Citation(s) in RCA: 123] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.
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Chayama K, Hayes CN, Hiraga N, Abe H, Tsuge M, Imamura M. Animal model for study of human hepatitis viruses. J Gastroenterol Hepatol 2011; 26:13-8. [PMID: 21175788 DOI: 10.1111/j.1440-1746.2010.06470.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Human hepatitis B virus (HBV) and hepatitis C virus (HCV) infect only chimpanzees and humans. Analysis of both viruses has long been hampered by the absence of a small animal model. The recent development of human hepatocyte chimeric mice has enabled us to carry out studies on viral replication and cellular changes induced by replication of human hepatitis viruses. Various therapeutic agents have also been tested using this model. In the present review, we summarize published studies using chimeric mice and discuss the merits and shortcomings of this model.
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Affiliation(s)
- Kazuaki Chayama
- Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Japan Liver Research Project Center, Hiroshima, Japan.
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Xie Y, Zhai J, Deng Q, Tiollais P, Wang Y, Zhao M. Entry of hepatitis B virus: mechanism and new therapeutic target. ACTA ACUST UNITED AC 2010; 58:301-7. [PMID: 20570056 DOI: 10.1016/j.patbio.2010.04.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Accepted: 04/12/2010] [Indexed: 12/21/2022]
Abstract
Entry of hepatitis B virus (HBV) into human hepatocytes constitutes the initial step in viral infection. The study of HBV entry had long been hampered by the lack of efficient cell culture systems and small animal models. The situation was greatly improved in the last decade with the development of HBV-infectible HepaRG cell line and primary Tupaia hepatocyte culture. Armed with these new tools, marked progresses have been achieved in the elucidation of the mechanism of HBV entry. Plenty of evidences indicate that the viral large surface protein (LHBs) is essential for HBV entry. Several regions in the PreS1 domain of LHBs have been verified to contribute directly to the viral attachment. In addition, a myristate moiety linked to the N-terminal glycine of PreS1 appears critical for HBV infectivity. Recently, the cysteine-rich antigenic loop of the S domain was identified as another crucial determinant for HBV infectivity. On the other hand, several cellular proteins were implicated in HBV attachment to hepatic cells, though definitive proofs are required in support to their functional involvement in HBV infection. Aiming to blocking viral entry, a couple of approaches based on acylated PreS1-derived peptides and short PreS1-binding peptides are currently under investigation, which have the potential to become novel antiviral therapeutics.
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Affiliation(s)
- Y Xie
- Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, China.
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HCV animal models: a journey of more than 30 years. Viruses 2009; 1:222-40. [PMID: 21994547 PMCID: PMC3185497 DOI: 10.3390/v1020222] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2009] [Revised: 08/05/2009] [Accepted: 08/18/2009] [Indexed: 12/13/2022] Open
Abstract
In the 1970s and 1980s it became increasingly clear that blood transfusions could induce a form of chronic hepatitis that could not be ascribed to any of the viruses known to cause liver inflammation. In 1989, the hepatitis C virus (HCV) was discovered and found to be the major causative agent of these infections. Because of its narrow tropism, the in vivo study of this virus was, especially in the early days, limited to the chimpanzee. In the past decade, several alternative animal models have been created. In this review we review these novel animal models and their contribution to our current understanding of the biology of HCV.
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Brezillon N, Kremsdorf D, Weiss MC. Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens. Dis Model Mech 2009; 1:113-30. [PMID: 19048074 DOI: 10.1242/dmm.000463] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
It has long been known that hepatocytes possess the potential to replicate through many cell generations because regeneration can be achieved in rodents after serial two-thirds hepatectomy. It has taken considerable time and effort to harness this potential, with liver regeneration models involving hepatocyte transplantation developing over the past 15 years. This review will describe the experiments that have established the models and methodology for liver repopulation, and the use of cells other than adult hepatocytes in liver repopulation, including hepatic cell lines and hematopoietic, cord blood, hepatic and embryonic stem cells. Emphasis will be placed on the characteristics of the models and how they can influence the outcome of the experiments. Finally, an account of the development of murine models that are competent to accept human hepatocytes is provided. In these models, liver deficiencies are induced in immunodeficient mice, where healthy human cells have a selective advantage. These mice with humanized livers provide a powerful new experimental tool for the study of human hepatotropic pathogens.
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Affiliation(s)
- Nicolas Brezillon
- INSERM, U845, Pathogenèse des Hépatites Virales B et Immunothérapie, Paris 75015, France
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Abstract
Hepatitis B virus (HBV) infection is a major worldwide healthy problem and now the virus and the virus-caused diseases have been known deeply. However, due to lack of a practical and convenient animal model, the study of HBV biology and the therapeutic development of HBV infection are still at a low level. As a common species used in laboratory, mice are studied most and the genetic and immune system are clearly understood. In this paper, we briefly describes the mouse models currently available in HBV.
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Zeira E, Manevitch A, Manevitch Z, Kedar E, Gropp M, Daudi N, Barsuk R, Harati M, Yotvat H, Troilo PJ, Griffiths TG, Pacchione SJ, Roden DF, Niu Z, Nussbaum O, Zamir G, Papo O, Hemo I, Lewis A, Galun E. Femtosecond laser: a new intradermal DNA delivery method for efficient, long‐term gene expression and genetic immunization. FASEB J 2007; 21:3522-33. [PMID: 17575264 DOI: 10.1096/fj.06-7528com] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.
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Affiliation(s)
- Evelyne Zeira
- The Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, P.O. Box 12000, Jerusalem, 91120 Israel
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34
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Kay A, Zoulim F. Hepatitis B virus genetic variability and evolution. Virus Res 2007; 127:164-76. [PMID: 17383765 DOI: 10.1016/j.virusres.2007.02.021] [Citation(s) in RCA: 208] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2006] [Revised: 01/12/2007] [Accepted: 02/22/2007] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus has been evolving gradually over a long period of time, resulting in a large amount of genetic diversity, despite the constraints imposed by the complex genetic organization of the viral genome. This diversity is partly due to virus/host interactions and partly due to parallel evolution in geographically distinct areas. Recombination also appears to be an important element in HBV evolution. Also, human intervention in the form of mass vaccination and antiviral treatment will reduce the burden of HBV-related liver disease but may also be accelerating evolution of the virus.
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Affiliation(s)
- Alan Kay
- INSERM, U871, Physiopathologie moléculaire et nouveaux traitements des hépatites virales, 151 cours A. Thomas, Lyon F-69424, France.
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35
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Dandri M, Volz T, Lutgehetmann M, Petersen J. Modeling infection with hepatitis B viruses in vivo. Future Virol 2006. [DOI: 10.2217/17460794.1.4.461] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Hepatitis B virus (HBV) is a human-specific liver pathogen whose viral cycle and mechanisms of pathogenesis are not yet fully understood. Along with invaluable infection studies in chimpanzees, avian and mammalian HBV-related viruses continue to offer ample opportunities for studies in their natural hosts. Yet, none of these hosts are commonly used laboratory animals; the lack of reliable in vitro infection systems and convenient animal models has severely hampered progress in HBV research. The need to perform studies in HBV-permissive hepatocytes has led researchers to create new, challenging human–mouse chimera infection models. The types of animal models currently available to perform infection studies with HBV are presented and discussed in this review.
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Affiliation(s)
- Maura Dandri
- University Hospital Hamburg, Department of Medicine, University of Hamburg, Eppendorf, Martinistr 52, D-20246 Hamburg, Germany
| | - Tassilo Volz
- University Hospital Hamburg, Department of Medicine, University of Hamburg, Eppendorf, Martinistr 52, D-20246 Hamburg, Germany
| | - Marc Lutgehetmann
- University Hospital Hamburg, Department of Medicine, University of Hamburg, Eppendorf, Martinistr 52, D-20246 Hamburg, Germany
| | - Jorg Petersen
- University Hospital Hamburg, Department of Medicine, University of Hamburg, Eppendorf, Martinistr 52, D-20246 Hamburg, Germany
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36
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Owada T, Matsubayashi K, Sakata H, Ihara H, Sato S, Ikebuchi K, Kato T, Azuma H, Ikeda H. Interaction between desialylated hepatitis B virus and asialoglycoprotein receptor on hepatocytes may be indispensable for viral binding and entry. J Viral Hepat 2006; 13:11-8. [PMID: 16364077 DOI: 10.1111/j.1365-2893.2005.00648.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.
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Affiliation(s)
- T Owada
- Hokkaido Red Cross Blood Center, Nishi-ku, Sapporo-shi, Hokkaido, Japan.
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37
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Abstract
The generation of small animal models, which preserve the ability for the generation of primary and memory immune responses of the engrafted human immune cells and in which a robust HIV-1 infection may occur, may enable the rapid screening, development and evaluation of HIV-1 protective vaccines and adjuvants. This manuscript reviews the existing mouse HIV-1 models used to study virologic, immunologic and pathogenic aspects of HIV-1 infection and disease and discusses their limitations and advantages, especially in the context of vaccine development, with special focus on the recently developed Trimera-HIV-1 animal model.
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38
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Dandri M, Volz TK, Lütgehetmann M, Petersen J. Animal models for the study of HBV replication and its variants. J Clin Virol 2005; 34 Suppl 1:S54-62. [PMID: 16461225 DOI: 10.1016/s1386-6532(05)80011-3] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Enormous progresses in hepatitis B virus research have been made through the identification of avian and mammalian HBV related viruses, which offer ample opportunities for studies in naturally occurring hosts. However, none of these natural hosts belongs to the commonly used laboratory animals, and the development of various mouse strains carrying HBV transgenes offered unique opportunities to investigate some mechanisms of viral pathogenesis. Furthermore, the need to perform infection studies in a system harbouring HBV-permissive hepatocytes has lately led researchers to create new challenging human mouse chimera models of HBV infection. In this review, we will overview the type of animal models currently available in hepadnavirus research.
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Affiliation(s)
- M Dandri
- Department of Medicine, University Hospital Eppendorf University of Hamburg, Martinistr 52, D-20246 Hamburg, Germany
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39
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Tsuge M, Hiraga N, Takaishi H, Noguchi C, Oga H, Imamura M, Takahashi S, Iwao E, Fujimoto Y, Ochi H, Chayama K, Tateno C, Yoshizato K. Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus. Hepatology 2005; 42:1046-54. [PMID: 16250045 DOI: 10.1002/hep.20892] [Citation(s) in RCA: 114] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Studies of hepatitis B virus (HBV) mutants have been hampered by the lack of a small animal model with long-term infection of cloned HBV. Using a mouse model in which liver cells were highly replaced with human hepatocytes that survived over a long time with mature human hepatocyte function, we performed transmission experiments of HBV. Human serum containing HBV and the virus produced in HepG2 cell lines that transiently or stably transfected with 1.4 genome length HBV DNA were inoculated. Genetically modified e-antigen-negative mutant strain also was produced and inoculated into the mouse model. A high-level (approximately 10(10) copies/mL) viremia was observed in mice inoculated with HBV-positive human serum samples. The level of viremia tended to be high in mice with a continuously high human hepatocyte replacement index. High levels and long-lasting viremia also were observed in mice injected with the in vitro generated HBV. The viremia continued up to 22 weeks until death or killing. Passage experiments showed that the serum of these mice contained infectious HBV. Genetically engineered hepatitis B e antigen-negative mutant clone also was shown to be infectious. Lamivudine effectively reduced the level of viremia in these infected mice. In conclusion, this mouse model of HBV infection is a useful tool for the study of HBV virology and evaluation of anti-HBV drugs. Our results indicate that HBeAg is dispensable for active viral production and transmission.
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Affiliation(s)
- Masataka Tsuge
- Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima-shi, Japan
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40
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Ayash-Rashkovsky M, Borkow G, Davis HL, Moss RB, Moss RB, Bartholomew R, Bentwich Z. Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection. FASEB J 2005; 19:1152-4. [PMID: 15833766 DOI: 10.1096/fj.04-3185fje] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
We have recently developed a novel small animal model for HIV-1 infection (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3184fje; doi:10.1096/fj.04-3184fje). The mice were successfully infected with HIV-1 for 4-6 wk with different clades of either T- or M-tropic isolates. HIV-1 infection was accompanied by rapid loss of human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation. HIV specific human humoral and cellular immune responses were observed in all HIV-1 infected animals. In the present study, HIV specific human immune responses, both humoral and cellular, were generated in noninfected Trimera mice, after their immunization with gp120-depleted HIV-1 antigen, presented by autologous human dendritic cells. Addition of CpG ODN to the antigen-pulsed DCs significantly enhanced (by 2- to 30-fold) the humoral and cellular HIV-1 specific immune responses. Only mice immunized with the HIV-1 immunogen and CpG were completely protected from infection with HIV-1 after challenge with high infection titers of the virus. This novel small animal model for HIV-1 infection may thus serve as an attractive platform for rapid testing of candidate HIV-1 vaccines and of adjuvants and may shorten the time needed for the development and final assessment of protective HIV-1 vaccines in human trials.
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41
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Ayash-Rashkovsky M, Bentwich Z, Arditti F, Friedman S, Reisner Y, Borkow G. A novel small animal model for HIV-1 infection. FASEB J 2005; 19:1149-51. [PMID: 15833767 DOI: 10.1096/fj.04-3184fje] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Lethally irradiated normal BALB/c mice, reconstituted with murine SCID bone marrow and engrafted with human PBMC (Trimera mice), were used to establish a novel murine model for HIV-1 infection. The Trimera mice were successfully infected with different clades and primary isolates of T- and M-tropic HIV-1, with the infection persisting in the animals for 4-6 wk. Rapid loss of the human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation accompanied the viral infection. All HIV-1 infected animals were able to generate both primary and secondary immune responses, including HIV specific human humoral and cellular responses. In addition to testing the efficacy of new antiviral compounds, this new murine HIV-1 model may be used for studying host-virus interactions and, most importantly, for screening and developing potential HIV-1 protective vaccines and adjuvants (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3185fje; doi:10.1096/fj.04-3185fje.).
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42
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Guha C, Lee SW, Chowdhury NR, Chowdhury JR. Cell culture models and animal models of viral hepatitis. Part II: hepatitis C. Lab Anim (NY) 2005; 34:39-47. [PMID: 15685191 DOI: 10.1038/laban0205-39] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2003] [Accepted: 12/16/2004] [Indexed: 12/25/2022]
Abstract
The lack of a preventive vaccine, coupled with common unresponsiveness to treatment and coinfection with HIV, has made HCV a major threat to public health. The authors review in vitro and in vivo models that are being used to study HCV and to develop new treatments and preventive measures.
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Affiliation(s)
- Chandan Guha
- Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA
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43
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Dandri M, Burda MR, Zuckerman DM, Wursthorn K, Matschl U, Pollok JM, Rogiers X, Gocht A, Köck J, Blum HE, von Weizsäcker F, Petersen J. Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes. J Hepatol 2005; 42:54-60. [PMID: 15629507 DOI: 10.1016/j.jhep.2004.09.021] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2004] [Revised: 09/09/2004] [Accepted: 09/17/2004] [Indexed: 01/18/2023]
Abstract
BACKGROUND/AIMS Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
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Affiliation(s)
- Maura Dandri
- Department of Medicine, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
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44
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Fellig Y, Almogy G, Galun E, Ketzinel-Gilad M. A hepatocellular carcinoma cell line producing mature hepatitis B viral particles. Biochem Biophys Res Commun 2004; 321:269-74. [PMID: 15358171 DOI: 10.1016/j.bbrc.2004.06.148] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2004] [Indexed: 12/12/2022]
Abstract
Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10II, exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10II cell genome. The presence of HBV in the FLC4A10II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10II, can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents.
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Affiliation(s)
- Yakov Fellig
- Department of Pathology, Hadassah University Hospital, Jerusalem, Israel
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45
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Guha C, Mohan S, Roy-Chowdhury N, Roy-Chowdhury J. Cell culture and animal models of viral hepatitis. Part I: hepatitis B. Lab Anim (NY) 2004; 33:37-46. [PMID: 15224117 DOI: 10.1038/laban0704-37] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2003] [Accepted: 02/25/2004] [Indexed: 12/19/2022]
Abstract
Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health, suggesting the need for research to develop new treatments to combat the disease. The authors review the available in vitro and in vivo models, including recently developed transgenic and chimeric mouse models.
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Affiliation(s)
- Chandan Guha
- Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA
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46
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N/A. N/A. Shijie Huaren Xiaohua Zazhi 2004; 12:1650-1655. [DOI: 10.11569/wcjd.v12.i7.1650] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/26/2023] Open
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47
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Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 2004; 8:769-76. [PMID: 14599810 DOI: 10.1016/s1525-0016(03)00244-2] [Citation(s) in RCA: 234] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Current therapies for chronic hepatitis B virus (HBV) infection are limited in their effect on viral gene expression and replication. Recent reports have shown that RNA interference can be induced in mammalian cells by short interfering RNA duplexes (siRNA). Here we studied the effects of an HBV-specific 21-bp siRNA targeted to the surface antigen region (HBsAg), where three major viral mRNAs overlap, on HBV gene expression and replication both in a cell culture system and in a mouse model for HBV replication. Transfection of siRNA into HepG2.2.15 cells, which constitutively produce HBV particles, caused a significant reduction in viral RNA production that was accompanied by a >80% drop in the secretion of viral HBsAg and HBeAg into the medium. The effect of RNAi was tested in vivo in a mouse model that we have developed for HBV infection, which entails hydrodynamic injection of a plasmid bearing the HBV genome into tail veins of mice. Injection of the HBV plasmid induces viral replication and generation of HBV viral particles detectable in the mouse sera. Co-injection of the HBV plasmid together with siRNA caused a significant inhibition in the level of viral transcripts, viral antigens, and viral DNA detected in the livers and sera of the treated mice relative to control animals. Results suggest that siRNA is capable of inhibiting HBV replication in vivo and thus may constitute a new therapeutic strategy for HBV infection.
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Affiliation(s)
- Hilla Giladi
- Goldyne Savad Institute of Gene Therapy, 91120, Jerusalem, Israel
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48
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N/A, 赵 文, 陈 耀, 王 宇. N/A. Shijie Huaren Xiaohua Zazhi 2004; 12:190-193. [DOI: 10.11569/wcjd.v12.i1.190] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 03/05/2023] Open
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49
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Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Shen ZH, Song YL, Cao AP. Inhibitory effect of oxymatrine on expression of hepatitis B virus in HBV transgenic mice. Shijie Huaren Xiaohua Zazhi 2004; 12:89-92. [DOI: 10.11569/wcjd.v12.i1.89] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the inhibitory effect of oxymatrine on HBsAg, HBeAg, and HBcAg expression in the liver tissue of HBV transgenic mice, and to further expound the mechanisms of oxymatrine anti-HBV.
METHODS: HBV transgenic mice models were established by microinjecting methods, and detected by HBV DNA integration and replication. Replicating HBV transgenic mice were divided into three groups: injected with normal saline (n = 9), 50 mg/kg (n = 8) and 100mg/kg (n = 9) oxymatrine intraperitonealy once a day for 30 d, respectively. After treatment, detection of HBsAg and HBeAg by ELISA, HBsAg and HBcAg by immunohistochemistry in the liver tissues was conducted.
RESULTS: Compared with group normal saline, HBsAg content in 50 mg/kg and 100 mg/kg oxymatrine decreased, but there was no statistic significance (F = 1.29, P>0.05). Compared with normal saline group, HBeAg content in 50 mg/kg and 100 mg/kg oxymatrine groups obviously decreased(F = 9.09, P < 0.01), but there was no obvious significance between 50 mg/kg and 100 mg/kg oxymatrine groups (F = 1.58, P> 0.05). The number of HbsAg-positive cells in the normal saline group, 50 mg/kg and 100 mg/kg oxymatrine had no changes in the liver tissues (x2 = 1.61, P> 0.05). The number of HBcAg-positive cells in the liver tissues was significantly lower in the group of 100 mg/kg oxymatrine than that in the group of normal saline (x2= 4.73, P < 0.05), but the number between normal saline group and 50 mg/kg oxymatrine group, and between the groups of 50 mg/kg and 100 mg/kg oxymatrine had no obvious significance.
CONCLUSION: There is an inhibitory effect of oxymatrine on expression of hepatitis B virus in HBV transgenic mice.
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Abstract
Potent inhibition of endogenous gene expression by RNA interference has been achieved by using sequence-specific posttranscriptional gene silencing through the action of small interfering RNA molecules (siRNA). In these reports, the natural function of genes could be deduced through the ensuing loss of function. Based on the extraordinary effectiveness in silencing endogenous genes, we wondered whether siRNA could be applied against viral replication in a hepatitis B virus (HBV) model using HBV-specific siRNA. To test this idea, HepG2 2.2.15, a human hepatoblastoma cell line that constitutively produces infectious HBV particles, was transfected with HBV-specific siRNAs and controls. HBV surface antigen (HBsAg) secretion into culture media was inhibited by 78%, 67%, and 42% with siRNA against the polyadenylation (PA), precore (PreC), and surface (S) regions, respectively, compared with controls as detected by enzyme-linked immunosorbent assay. After exposure to HBVPA siRNA, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 72%, and levels of HBV RNA containing the polyadenylation signal sequence were suppressed by 86%, as detected by RNase protection assay. Levels of HBV core-associated DNA, a replication intermediate, also decreased by 71%. Immunocytochemistry revealed that 30% to 40% of the cells transfected with HBVPA siRNA were completely negative for detectable HBsAg levels. Controls consisting of treatment with HBV-specific siRNA alone, lipofection reagent alone, or random double-stranded RNA (dsRNA) lipofection complex failed to decrease HBV surface antigen, HBV messenger RNA (mRNA), or core-associated HBV-DNA levels. In conclusion, siRNA inhibits hepatitis B viral replication in a cell culture system. Future studies are needed to explore the specific delivery of siRNA to liver cells in vivo and the applicability of this approach.
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Affiliation(s)
- Masayoshi Konishi
- Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1845, USA
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