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Keywords:

  • hepatitis B virus;
  • hepatitis C virus;
  • uPA/scid mouse model

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are pathogens that cause chronic infection in humans. There are 360 million and 170 million people infected worldwide with HBV or HCV, respectively.1,2 Infected individuals develop acute hepatitis, chronic hepatitis and liver cirrhosis. The viruses are also important causative agents of hepatocellular carcinoma, especially in the Asia–Pacific region.3 Study of the biology and development of therapies for each virus has long been hampered by the lack of a small animal model that supports hepatitis virus infection. This is probably as a result of the lack of receptor molecules necessary for viral infection in animal liver cells.

Transgenic mice that express over-length HBV-DNA export viral particles into the serum,4 and such animals can be used to evaluate antiviral agents,5–7 as well as HBV-targeted siRNA8. However, the virus life cycle is not established in this model, and it is inappropriate for studying drug-resistant HBV strains. Accordingly, researchers attempted to transplant human hepatocytes into mice. The development of the trimera mouse was one such attempt, in which human hepatocytes were transplanted under the kidney capsule of immune-deficient mice after lethal irradiation.9,10 However, the number of hepatocytes that could survive on the kidney capsule was small, and normal liver architecture was not present. Although 85% of HBV-inoculated animals developed HBV viremia, the titer was less than 105 virus particles or IU/mL.9 Similarly, 85% of HCV-inoculated animals also developed viremia,10 but the level of the viremia only reached 105/mL. Thus, the advent of human hepatocyte transplanted uPA/scid mice has provided the first really useful model for acute and chronic infections of human hepatitis virus.

Human liver cell transplanted uPA/scid mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

Transgenic mice in which the urokinase gene is driven by the human albumin promoter/enhancer were developed and shown to have accelerated hepatocyte death and consequent chronic stimulation of hepatocyte growth.11 Transplanted rat hepatocytes proliferated and repopulated injured livers in immunodeficient uPA mice, which were produced by mating uPA transgenic mice with scid mice.12 Human hepatocytes were then transplanted into uPA/scid mice; these cells proliferated and replaced the apoptotic mice liver cells (Fig. 1).

image

Figure 1. Generation of human hepatocyte chimeric mice and hepatitis virus infection model. A uPA/scid mouse was created by mating uPA transgenic mouse and scid mouse. Human hepatocytes obtained by surgical resection or donation were transplanted to newborn mice. The chimeric mice can be infected with hepatitis B virus (HBV) or hepatitis C (HBC) virus by injecting human serum containing these viruses. Alternatively, the mice can be infected by HBV13 or HCV14 created in cell culture or by injecting HCV RNA into the mouse liver.15

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Such human hepatocyte chimeric mice have been shown to be susceptible to both HBV16 and HCV17 infections. Repopulation levels by human hepatocytes have been estimated by measuring human albumin levels in mouse serum. Replication levels of both HBV13 and HCV17 were higher in mice in which the repopulation index was higher. A unique attempt to remove mouse residual liver cells with the herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system failed to result in a higher repopulation rate as a result of damage to the transplanted human hepatocyte caused by bystander effects.18 Despite this, mice with livers that have been highly repopulated with human hepatocytes are susceptible to infection with both HBV and HCV, and as such comprised the most effective small animal model for chronic hepatitis so far developed.19,20 An example of a highly repopulated mouse liver that we are using in experiments is shown in Figure 2.

image

Figure 2. Representative uPA/scid mouse livers repopulated by human hepatocytes. (a) Mouse liver almost completely repopulated by human hepatocytes. Only a small portion of mouse hepatocytes are shown by arrows. (b) Microscopic figure of the mouse liver. M and H indicate regions consisting of mouse and human hepatocytes, respectively (Hematoxylin–eosin staining, magnification: ×100). (c) Microscopic figure of the mouse liver stained with antibody directed against human serum albumin.

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Highly repopulated mice have been shown to be a valuable model for the study of drug metabolism.21–29 Advances in technology for human hepatocyte transplantation have enabled serial passage of human hepatocytes in uPA/scid mice and have been shown to retain infectivity for HBV.30

This mouse model and other animal models for the study of hepatitis viruses have been summarized in reviews by Meuleman and Leroux-Roels,31 Dandri et al.,32,33 Barth et al.,34 and Kneteman and Toso.35 The present review will focus on key issues and updated information.

Study of hepatitis B virus infection using human hepatocyte chimeric mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

Since the initial reports of successful transmission of HBV to human hepatocyte chimeric mice in 2001 and 2004,16,27 several researchers have reported transmission of HBV into similar mice.13,36,37 In these studies, passage experiments studies show that HBV replicating in mice retain infectivity.13,36 Further, the presence of viral proteins has been shown immunohistochemically in human hepatocytes transplanted into mouse livers, but these are not present in mouse hepatocytes.13,36,37 Formation of viral particles in infected mouse livers can be shown by electron microscopy.36,37 Genetically engineered viruses lacking HBe-antigen have also been shown to infect chimeric mice, proving that e antigen is dispensable for viral infection and replication.13 In contrast, HBx protein has been shown to be indispensable for viral replication.38 Transcomplementation of HBx protein with hydrodynamic injection restored HBV infectivity in mice. Interestingly, all revertant viruses show a restored ability to express HBx.38

By infecting chimeric mice with genotype A, B and C, differing proliferative capacity has been shown between HBV genotypes.37 In mice infected for a relatively short time, there are no morphological changes in HBV infected mice livers in studies.13,36 In contrast, the occurrence of liver cell damage has been reported after long-term infection of chimeric mice with HBV39 or with specific strains of HBV;40 these findings are consistent with direct cytopathic effects of HBV under certain conditions.

The biological properties of a newly identified unique strain of HBV, genotype G, which replicates only in the presence of another genotype, were confirmed using the chimeric mouse.41 Infectivity of another novel HBV strain, identified from a Japanese patient, that is divergent from known human and ape HBV has also been confirmed.42 Titration of HBV infectivity, which previously could only be carried out using chimpanzees, can be carried out effectively using chimeric mice.43

Taking advantage of the absence of human immune cells in the chimeric mice, Noguchi et al.44 showed that hypermutation of HBV increases in human hepatocytes under interferon treatment. Dandri et al. measured viral half-life in human and chimeric mice repopulated with wooly monkey hepatocytes.45 The results clearly showed that viral half-life is shortened by immunological mechanisms in humans with low viral levels, but not in chimeric mice where functional immunity is absent. Hiraga et al.46 showed an absence of interference between HBV and HCV.

Evaluation of therapeutic agents is the most important role for this mouse model. Tsuge et al.13 assessed the effect of interferon and lamivudine using chimeric mice. Similarly, Dandri et al.47 showed the effects of adefovir using uPA/scid mice repopulated with tupaia hepatocytes, which also support replication of human HBV. Oga et al.48 identified a novel lamivudine-resistant variant that has an amino acid substitution outside of the YMDD motif. They showed that lamivudine was ineffective against the novel mutant strain. It is thus apparent that this mouse/human liver chimeric model is ideal to study the susceptibility of mutant strains to various drugs, because mutant viruses can easily be made and infected into chimeric mice.13 The model has also been utilized to evaluate viral entry inhibitors derived from the large envelope protein.49

Study of hepatitis C virus using human hepatocyte chimeric mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

As observed in studies on HBV, HCV infection efficiency was poor and levels of viremia were low in mice where the repopulation rate of the mouse liver with human hepatocyte was low.17,50 As shown in Figure 3, human albumin levels in mouse serum were significantly higher in mice in which measurable viremia developed (Hiraga et al. unpublished data). Recent studies have therefore been carried out using highly repopulated mice. The usefulness of a newly developed HCV assay,51 and infectivity of a newly identified intergenotypic recombinant strain,52 have been reported using the chimeric mice.

image

Figure 3. Human albumin levels in mice used in the hepatitis C virus (HCV) infection experiments. A total of 54 mice were injected with HCV positive serum samples containing 5 × 105 virus particles. A total of 24 mice became persistently positive for HCV-RNA, but 30 mice did not. Serum human albumin levels 2 weeks after human hepatocyte transplantation were compared between infected and non-infected mice.

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Using the remarkable replication ability of the JFH1 genotype 2a strain,53 infectivity of JFH1 or intergenotypic chimeric viral particles, previously shown in cell culture, has now been shown to be infectious in chimeric mice.54–56 Infectivity of viruses that were replicated in chimeric mice in cell culture has also been shown, and virus fitness has been studied.55,56 The role of the HCV core+1 open reading frame and core cis-acting RNA elements has also been examined using the chimeric virus.57 These elegant studies have the limitation that the non-structural part of the virus is limited to that of JFH1. Hiraga et al.14 have shown that infectious clones of genotype 1a and JFH1 can be infected with direct injection of in vitro transcribed RNA into the mouse liver.14 Similarly, Kimura et al.15 reported the establishment of infectious clones of genotype 1b and ablation of RNA polymerase by site-directed mutagenesis abolish infectivity. These infectious clones will be useful for the study of drug-resistant strains.

The model of HCV infection has also been used to show that infection of the virus can be prevented by antibodies against CD81,58 polyclonal human immunoglobulin directed to a similar strain,59 and amphipathic DNA polymers.60 Notably, the presence of broadly neutralizing antibodies to HCV that protect against heterologous viral infection has been reported, suggesting the possibility of a prophylactic vaccine against HCV.61

With respect to evasion of the virus against the innate immune response, altered intrahepatic expression profiles in the early phase of infection is of particular interest. The chimeric mice model is ideal for such studies; cross-hybridization of mouse and human can be avoided by careful experimental procedures.62 Microarray analysis of livers of HCV infected and non-infected mice showed transcriptional activation of genes related to innate immune response, lipid metabolism, endoplasmic reticulum (ER) stress and apoptosis in HCV-infected mice.63,64 The HCV infected mouse model is particularly useful for the study of newly developed HCV agents. The effect of recently developed chemicals and a unique therapy using intrahepatic lymphocytes have been shown using this model (Table 1). However, none of these therapies have yet been able to completely eradicate HCV from mice. It is noteworthy that ultra-rapid cardiotoxicity has been reported with the protease inhibitor BILN 2061 in the uPA/scid mice, but not in scid mice, implicating involvement of the uPA transgene.72 Care should therefore be taken in interpreting the results obtained by this model.

Table 1.  New therapeutic strategies tested by human hepatocyte chimeric mice
nDrug or cellStrategyReference
1Interferon alpha 2bActivation of antiviral genesKneteman et al.65
BILN-2061NS3-4A protease inhibition 
HCV371NS5B polymerase inhibition 
2Modified BIDInduction of apoptosisHsu et al.66
3Serine palmitoyltransferase inhibitorDisruption of lipid raftUmehara et al.67
4Lymphoblastoid interferon alphaActivation of antiviral genesHiraga et al.14
5Amphipathic DNA polymersBlocking viral entryMatsumura et al.60
6Sec-butyl-analogue of HCV-371NS5B polymerase inhibitionLaPorte et al.68
7HCV796NS5B polymerase inhibitionKneteman et al.69
8Liver allograft-derived lymphocyteAdoptive immunotherapyOhira et al.70
9TelaprevirNS3-4A protease inhibitionKamiya et al.71

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

Development of a small animal model using human hepatocyte chimeric mice has enabled us to study key aspects of HBV and HCV biology. The characteristic feature of the absence of human immune cells is suitable for studying viral replication and observing changes occurring in liver cells during viral infection, such as the innate immune response and cellular stress and metabolic responses. The model is also useful for studying the effect of drugs without the influence of cytokines and cytotoxic T lymphocytes. Nonetheless, the model is insufficient to study carcinogenesis of hepatitis viruses, because non-parenchymal cells in mouse liver are of mouse origin and do not support inflammation and fibrosis, which are probably closely related to carcinogenesis. The lack of human immune cells also limits the study of inflammation and immunity. Furthermore, the availability of human hepatocytes is limited. Despite these limitations, the current model shows great potential as a mouse model for the study of hepatitis viruses. Development of a small animal model with or without human immunity using stem cells or iPS cells would be an ideal model in the future.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References

This work was supported in part by Grants-in-Aid for scientific research and development from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Government of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Human liver cell transplanted uPA/scid mice
  5. Study of hepatitis B virus infection using human hepatocyte chimeric mice
  6. Study of hepatitis C virus using human hepatocyte chimeric mice
  7. Conclusion
  8. Acknowledgments
  9. References