Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes

Journal of Hepatology 42 (2005) 54–60 www.elsevier.com/locate/jhep

Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes* Maura Dandri1,†, Martin R. Burda1,†, David M. Zuckerman2, Karsten Wursthorn1, Urte Matschl1, Joerg M. Pollok3, Xavier Rogiers3, Andreas Gocht4, Josef Ko¨ck5, Hubert E. Blum5, Fritz von Weizsa¨cker5, Joerg Petersen1,2,* 2

1 Department of Medicine, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Heinrich Pette Institute for Experimental Virology, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 3 Department of Hepatobiliary Surgery, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 4 Institute of Pathology, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 5 Department of Medicine, University of Freiburg, Germany

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 alpha1antitrypsin 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. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Xenotransplantation; UPA/RAG-2 mice; Cryopreservation; HBV; Woolly monkey; cccDNA; Antiviral therapy

Received 27 July 2004; received in revised form 9 September 2004; accepted 17 September 2004; available online 19 October 2004 * This work was presented in parts at the 53rd Annual Meeting of the AASLD in Boston 2002 and in Elmau Conference in 2003 supported by the German National Hep-Net. * Corresponding author. Address: Department of Medicine, University of Hamburg, Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: C49 40 42803 3926; fax: C49 40 42803 8065. E-mail address: [email protected] (J. Petersen). Abbreviations AAT, alpha-1-antitrypsin; ccc, covalently closed circular; HBsAg, hepatitis B surface antigen; PTH, primary tupaia hepatocytes; PVDF, polyvinylidene difluoride; RAG-2, recombination activation gene-2; uPA, uroplasminogen activator; WM, woolly monkey. † Both authors contributed equally to this work. 0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.09.021

M. Dandri et al. / Journal of Hepatology 42 (2005) 54–60

1. Introduction Hepatitis B virus (HBV) infection is a major public health problem and an important cause of infectious disease mortality worldwide. Approximately 2 billion people have serologic evidence of past or present HBV infection, and 350 million people are chronically infected. Each year over 1 million people die from HBV-related chronic liver disease, including cirrhosis and hepatocellular carcinoma [1]. The understanding of the complete viral life cycle, as well as the development of more effective antiviral agents aiming at the eradication of the virus from chronic carriers, have been hampered by the lack of efficient in vitro infection systems and suitable animal models. Chimpanzees can be efficiently infected with HBV, but evaluation of antiviral drugs in chimpanzees, or other high primates, is limited for ethical and financial reasons. The study of HBVrelated viruses in woodchucks and ducks has improved our knowledge on hepatitis B virus replication mechanisms to great extend. However, woodchucks are difficult to handle in laboratories and hepadnavirus infection in birds is generally not associated with liver disease and hepatocellular carcinoma [2]. Though infectious hepatitis B virus can be produced in transgenic mice [3], the full viral life cycle, including uptake and elimination of the virus by antiviral drugs, cannot be fully investigated in this system, since mice are not susceptible to HBV infection and the virus is produced from a chromosomically integrated copy of the viral genome. Therefore, access to a model containing HBV-permissive hepatocytes would offer better opportunities to investigate all steps of viral infection and to address more meaningful testing of antiviral agents. The Asian tree shrew Tupaia belangeri, a squirrel-like animal phylogenetically related to primates [4], was found to be susceptible to HBV infection [5]. Nevertheless, inoculation with HBV causes only a transient infection in tree shrews and the level of gene expression and viral replication in the liver of these animals are rather low. Primary tupaia hepatocytes (PTH) cultures were also found to be reproducibly susceptible to infection with HBV and woolly monkey hepatitis B virus (WM-HBV) [5–9]. However, similar to the situation with cultured human hepatocytes, PTH are difficult to maintain in culture over prolonged periods of time and become quickly nonpermissive for HBV/WM-HBV after plating. In recent years, various models have been developed utilizing human liver tissues or isolated hepatocytes for transplantation into immunodeficient mice [10–13]. The discovery of a liver-toxic phenotype in urokinase-type plasminogen activator (uPA) transgenic mice led to the development of a liver repopulation model, in which hepatocyte-targeted over-expression of the albumin-uPA transgene leads to the death of transgene carrying hepatocytes resulting in a growth advantage for transplanted cells [14]. Mice transplanted with human hepatocytes were shown to be suitable for HBV infection studies in

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immunodeficient mice [10,12]. uPA/RAG-2 mice lack mature B and T lymphocytes due to a deletion in the recombination activation gene-2 [15]. Unfortunately, the shortage of usable primary human hepatocytes is a major limitation of these transplantation models [16]. Therefore, we explored here the potential of tupaia hepatocytes to establish a more accessible HBVinfection model in uPA/RAG-2 mice.

2. Experimental procedures 2.1. Animals uPA transgenic mice and RAG-2 knockout mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and Taconic Farms (Germantown, NY, USA), respectively. Animals were housed and maintained under specific pathogen-free conditions in accordance with institutional guidelines under approved protocols. The correct genotypes of the uPA/RAG-2 mice were determined by polymerase chain reaction (PCR) [10,16]. Asian tree shrews (Tupaia belangeri) were obtained from the German Primate Center in Go¨ttingen, Germany, and maintained in the animal facility of the University of Freiburg.

2.2. Preparation of primary tupaia hepatocytes PTH were isolated by a collagenase perfusion method [7]. Isolated hepatocytes were immediately used for transplantation or further processed for cryopreservation as reported [17]. Frozen cells were stored for several months in liquid nitrogen. For cell thawing, vials were briefly placed in a 37 8C water bath and then on ice. RPMI culture medium (GibcoBRL, Karlsruhe, Germany) was added to the cells using a multi-step protocol [17]. Viability was determined by trypan blue exclusion. 5!105 viable tupaia hepatocytes suspended in 50 ml phosphate-buffered saline (PBS) were transplanted into 13–23 day-old uPAC/K/RAG-2K/K mice by intrasplenic injection.

2.3. HBV/WM-HBV infection and antiviral studies HBV-positive serum (1!108 HBV-DNA genome equivalents/ml) was obtained from an HBsAg-positive chronic carrier (genotype D) (see also Table 2). Woolly monkey serum contained 1!109 WM-HBV DNA genome equivalents/ml and was a kind gift from M. Nassal (University of Freiburg, Germany). Serum samples were stored at K70 8C. Mice were injected intra-peritoneally with 20 ml of HBV or WM-HBV positive serum. Engrafted mice with stable viral titres S5!105 HBV or WM-HBV genome equivalents were used for antiviral studies. 3 mg of adefovir dipivoxil per day was orally administrated to the mice for several weeks as described in the results.

2.4. Detection of alpha-1-antitrypsin and viral envelope proteins in mouse sera Serum samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. The blots were probed with a mouse monoclonal antibody against human alpha-1-antitrypsin (AAT) (1:2000 dilution; Biotrend, Koeln, Germany), which cross-reacts with tupaia alpha-1-antitrypsin and does not cross-react with mouse proteins. Specific binding was detected by chemiluminescence as previously reported [10]. For semi-quantitative determination of the relative amount of AAT in transplanted mice, artificial mixtures of tupaia and mouse serum were analysed along with the samples. Hepatitis B surface antigen (HBsAg) was measured by enzyme-linked immunosorbent assay (ELISA) [10].

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2.5. Detection and quantification of HBV and WM-HBV DNA in uPA/RAG-2 mice Viral DNA was extracted from serum samples and liver tissues using the QIAamp Blood Kit (Qiagen, Hilden, Germany) and the MasterPure DNA purification kit (Epicentre), respectively. Prior to cccDNA amplification, aliquots of each DNA sample were digested with plasmid-safe DNase. Real-time PCR was performed in a LightCycler (Roche, Basel, Switzerland) as described [18]. As control, known amounts of cloned HBV and WM-HBV DNA were amplified in parallel to establish a standard curve for quantification. The tissue values were normalized against the cellular DNA content by the beta-globulin assay (Roche, Basel, Switzerland). To determine the relative amount of tupaia cells within the mouse liver, the histological data were utilized as described below.

2.6. Histological studies Serial formalin fixed sections of transplanted and non-transplanted uPA/RAG-2 mouse livers were examined by hematoxylin-eosin (HE) staining or by immunohistochemistry. For immunohistochemistry either a monoclonal antibody directed against human alpha-1-antitrypsin (AAT) (Biotrend, Koeln, Germany), or a rabbit HBcAg antiserum, or a goat HBsAg antiserum (DAKO Diagnostika, Hamburg, Germany) were used. Sections were rehydrated, incubated with the individual antibodies (diluted 1:1; 1:250; or 1:500 in PBS containing 5% goat serum, respectively), and then treated with the specific biotinylated secondary antibodies (Vectastain ABC kit, Burlingame, CA, USA). To estimate the percentage of tupaia hepatocytes in mouse liver, four sections per liver lobe were cut from five lobes (20 sections per mouse) and stained with AAT-antibody. Sections were examined microscopically and the percentage of AAT-positive stained hepatocytes was calculated.

3. Results 3.1. Evaluation of xenogenic hepatocyte engraftment in mice

Table 1 Efficiency of hepatocyte engraftment into uPA/RAG-2 mice Tupaia livers

Hepatocyte source

Viability (%)

N. Transplanted mice

Survival ratio

N. Engrafted mice (AATR10%)

4 2

Fresh Cryopr.

90% 75%

58 40

85% 80%

40/50 23/32

Viability means the viability of PTH after isolation. Survival ratio is the ratio of mice surviving the transplantation procedure to the number of transplanted mice. Mice with a minimum level of 10% tupaia alpha-1antitrypsin (AAT) in the serum were regarded as successfully engrafted.

mouse serum (Fig. 1A). To evaluate the ratio of tupaia versus mouse AAT, tupaia and mouse serum mixtures were also immunoblotted. PTH engraftment was successful (AAT S10%) in approximately 80 and 70% of the mice that survived the transplantation with fresh or cryopreserved PTH, respectively (Table 1). AAT specific signals were first detectable 2 weeks after transplantation and reached steady state levels within 4–6 weeks (Fig. 1B). Long-term repopulation studies were performed with six mice showing AAT levels S50%. Production of AAT remained stable in all mice during the observation time of up to 1 year. A representative time course for AAT expression after transplantation is shown in Fig. 1B. To demonstrate repopulation of uPA/RAG-2 mouse livers with PTH, animals were sacrificed 2 months after transplantation and analyzed histologically. As shown in Fig. 2, HE staining and immunohistochemistry revealed broad areas of repopulation with large PTH nodules arranged in typical cord-like structures. Within xenogenic nodules, hepatocyte cytoplasm and nuclei appeared histologically normal compared with

Freshly isolated or cryopreserved PTH with S70% viability were used for transplantation experiments. To determine the extent of engraftment, mice were screened for the production of tupaia alpha-1-antitrypsin (AAT) 2–3 weeks after transplantation. AAT profiles were analyzed by immunoblotting using a monoclonal antibody that allows the specific identification of human and tupaia AAT in

Fig. 1. Secretion of alpha-1-antitrypsin in the serum of uPA/RAG-2 mice. Tupaia alpha-1-antitrypsin (AAT) was specifically detected in the serum of PTH transplanted mice by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and immunoblotting. (A) Production of AAT in six uPA/RAG-2 mouse sera 3 weeks after transplantation. (B) Levels of AAT detected in one mouse over a period of 1 year. The relative amount of AAT in engrafted mice was quantified by loading in parallel defined mixtures of tupaia serum in mouse serum; percentage (%vol./vol.); percentage of tupaia serum is given. All serum samples were diluted 1:50 before loading.

Fig. 2. Histological localization of tupaia hepatocytes in uPA/RAG-2 mice 8 weeks after transplantation. Differentiation of tupaia and mouse tissue by hematoxylin/eosin staining (A, B). Detection of AAT in two repopulated uPA/RAG-2 mouse livers (C, D) by staining with an alpha1-antitrypsin antiserum. (A: !20; B: 200, C, D: !50). [This figure appears in colour on the web.]

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Table 2 Establishment of HBV or WM-HBV infection in transplanted mice Serum samples

Viral type

Viral titre (GE/ml serum)

Infection in mice

Viral titre in mice (GE/ml serum)

HBV patient HBV patient Woolly monkey Woolly monkey HBV passage serum WM-HBV passage serum

D D WM-HBV WM-HBV D WM-HBV

1!108 1!108 1!109 1!109 5.4!107 8!107

2/3C3/3a 0/2 not-Txb 3/4C2/2a 0/2 not-Txb 2/2 2/2

5!105 to 8.2!107 !1!103 3!106 to 8!107 !1!103 5 and 6!106 3.2 and 3.7!107

Viral titres were determined by real time PCR. a Infection experiments performed for antiviral studies. b Infection experiments performed in not transplanted mice.

the surrounding mouse liver. AAT monoclonal antibody intensely stained control tupaia liver but did not cross-react with non-transplanted mouse liver (data not shown). The extent of mouse liver repopulation ranged between 30 and 80% and correlated with the AAT titres observed in serum (data not shown). Since only 5!105 PTH had been transplanted into each animal, a considerable degree of expansion of transplanted cells, both freshly isolated and cryopreserved, must have occurred. 3.2. Establishment of productive HBV and WM-HBV infection in transplanted uPA/RAG-2 mice Four weeks after transplantation, 3 uPA/RAG-2 mice with AAT levels S50% were injected with HBV-positive human serum. Mouse serum samples were collected between 2 and 50 weeks after inoculation and analyzed for the presence of surface antigen (HBsAg) by ELISA and HBV DNA by real-time PCR. As summarized in Table 2, two out of three transplanted mice became HBsAg positive 12 weeks after inoculation, while one animal remained negative throughout the observation period (Fig. 3A). High levels of HBV DNA (5.4!107 and 8.2!107 copies/ml) could be found by real time PCR in the two HBsAg positive mice and viral titres remained stable for the entire observation time (6 months). In a second set of experiments, WM-HBV positive serum was used. Three out of four transplanted mice inoculated with WM-HBV became HBsAg positive within 6 weeks (Fig. 3B) and reached viral titres between 3!106–8! 107 WM-HBV DNA/ml serum within 3 months of infection. Viral DNA and HBsAg were not detected in non-transplanted littermates that had been also inoculated using the same protocol (Table 2). The establishment of productive HBV or WM-HBV infection in transplanted uPA/RAG-2 mice was also determined by immunohistochemistry. Mouse liver sections randomly chosen from different lobes were stained for HBcAg and HBsAg. Six months after HBV or WM-HBV infection, HBcAg positive (Fig. 4A) and HBsAg positive (Fig. 4B and C) hepatocytes could be detected throughout the repopulated areas, while sections of not transplanted but HBV injected mice did not show any staining (Fig. 4D).

To determine whether infectious viral particles were produced in mice, we injected 20 ml of serum (w1!106 HBV DNA copies) obtained from a HBV-positive mice, into two naive chimeric mice. Serum samples were collected every other week starting 2 weeks after injection. Twelve weeks after inoculation, HBsAg became positive by ELISA in both mice (S/NZ16). Mice reached viral titres of 5 and 6!106 HBV DNA/ml 16 weeks post-inoculation, demonstrating a complete HBV life cycle in this mouse model (Table 2). Sequence analysis of the HBV genome in the original human serum, as well as in the infected mice both before and after viral passage, confirmed the presence of a predominant viral population (genotype D), with no obvious adaptive mutations found in infected mice (data not shown). Serum passage experiments were also performed by injecting WM-HBV-positive mouse serum into two additional chimeric mice. HBsAg was detected by ELISA in

Fig. 3. Detection of hepatitis B surface antigen in the sera of mice by ELISA. HBsAg was measured using the HBsAg Axsym Test (Abbott, Wiesbaden, Germany) using 1:200 dilution of the mouse sera. (A) Detection of HBsAg in sera of mice that had been inoculated with HBV 4 weeks after transplantation with PTH. (B) Detection of HBsAg in the sera of mice transplanted with PTH and inoculated with WM-HBV. S/N: positive versus negative sample ratios.

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3!103 WM-HBV DNA copies per cell, respectively. This corresponds to a WM-HBV total DNA/cccDNA ratio of 112 and 138. Total intracellular and cccDNA amounts were also analysed in liver tissues obtained from one HBV-positive mouse that had been sacrificed 6 months after infection. cccDNA levels (35 molecules per tupaia cell) and intracellular total viral DNA (3.8!103) were in the same range as in WM-HBV-infected mice (Table 3). 3.4. Adefovir dipivoxil inhibits viral DNA synthesis in repopulated uPA/RAG-2 mouse livers

Fig. 4. Histological localization of HBV and WM-HBV infected tupaia hepatocytes in uPA/RAG-2 mouse livers 6-month after transplantation. (A) Detection of HBcAg in HBV infected mouse liver by staining with an HBcAg antiserum. The darker appearing mouse hepatocytes on the left do not show any staining. Detection of HBsAg in HBV (B) and WMHBV (C) infected mouse livers by staining with HBsAg antiserum. (D) Liver tissue from a non-transplanted but HBV injected uPA/RAG-2 mouse showed no HBsAg reactivity (A, B, C, D: !200).

both mice within 6 weeks of inoculation and reached viral titres of up to 3.7!107 WM-HBV DNA/ml within 3 months. It is worth noting that the kinetics of infection were not changed both for HBV and WM-HBV that had been passaged through the mice, which is consistent with the apparent lack of adaptive mutations. 3.3. Detection of intracellular total HBV or WM-HBV DNA and cccDNA in infected mouse livers As shown recently in humans with chronic HBV infection, total intracellular and cccDNA in hepatocytes can be accurately quantified using real-time PCR [18]. We applied this method to mice infected with WM-HBV and HBV. To determine the presence of cccDNA in mice, we first used two transplanted and infected mice containing 10 and 4!106 WM-HBV DNA copies/ml serum, respectively. The total cellular DNA content in the assay was determined by optical density, while the proportion of tupaia cells within the mouse liver was assayed by histological analysis (see methods). Quantification with real time PCR revealed the presence of 106 and 24 cccDNA copies per tupaia liver cell, respectively, in the two WM-HBV infected animals (Table 3). The intracellular viral titres were 12!103 and

To demonstrate the usefulness of the chimeric tupaia/ mouse system for antiviral studies, tupaia repopulated mice were de novo infected with either HBV or WM-HBV infectious serum. As shown in Fig. 5, three HBV infected mice (panel A) and two WM-HBV infected animals (panel B) were treated with adefovir dipivoxil. A two-log reduction was observed in all HBV or WM-HBV infected mice within 6 or 10 weeks of treatment, respectively. Two WM-HBV infected untreated mice served as control. As shown in panel B, WM-HBV titres returned to w5!107 within 3 weeks of drug withdrawal, thus demonstrating the specific antiviral effect of adefovir dipivoxil.

4. Discussion We showed that primary tupaia hepatocytes are able to engraft xenogenic mouse livers with high efficiency. Using the previously described hemizygous uPA/RAG-2 mice [10] it was possible to achieve repopulation levels (up to 80%) comparable to the values found after transplantation of woodchuck hepatocytes [19] or other rodent hepatocytes [20,21]. This is substantially higher than the repopulation levels achieved with human hepatocytes [10]. One of the reasons for the different efficacies may be that human tissues are first flushed with a preserving solution and then perfused in vitro, while the liver of laboratory animals is perfused in situ without intermediate steps which may have a negative impact on engraftment or expansion capacity of the transplanted hepatocytes. It is also possible that cross-talk with non-parenchymal cells, matrix and growth factors in mice is more efficient for PTH compared to human hepatocytes, when transplanted and endogenously

Table 3 WM-HBV and HBV DNA levels in infected mouse livers Animal

WHV/HBV

Viral genome equivalents (!106)/ml serumGSD

ccc-DNA/cellGSD

Total viral DNA/cell (!103)GSD

Total/ccc

613 620 498

WHV WHV HBV

10.2G1.6 4.3G0.7 54G1.9

106.6G5.3 24.7G2.1 35.6G2.4

11.8G2.8 3.3G0.5 3.8G0.8

112 138 107

Serum and intracellular total viral DNA contents, as well as the intracellular cccDNA contents were determined by real time PCR. The DNA content of each sample was determined in five independent PCR reactions. The standard deviation (SD) is given.

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Fig. 5. Adefovir dipivoxil reduces viral titres of HBV and WM-HBV infected mice. A, Strong HBV DNA reduction in serum of three PTH engrafted mice after 30 days of adefovir treatment. B, Two mice out of four that had been transplanted with PTH and infected with WM-HBV were treated with adefovir. Arrows indicate when antiviral treatment was started; cessation of treatment is indicated by (?).

recombined mouse hepatocytes compete in the diseased mouse liver [14,20]. Our experiments show that uPA/RAG-2 mice repopulated with tupaia hepatocytes can be infected both with WM-HBV and HBV-positive human serum, confirming the results from in vitro cultivated PTH [7]. Successfully infected mice developed prolonged viral infection associated with high viral titres. Furthermore, our serum passage experiments prove that PTH support the full viral replication cycle. WM-HBsAg was already detectable at 6 weeks postinoculation, whereas HBV-HBsAg started showing positive reactions in the ELISA at 12 weeks post-inoculation. The longer lag time for productive HBV infection may be due to the fact that the evolutionary differences between the natural host of WM-HBV, the woolly monkey, and tupaia are smaller than between tupaia and humans [22]. Therefore, virus/host interactions between WM-HBV and PTH may be more efficient than the interactions of human HBV with tupaia cells. Though a lower replication rate of HBV compared to WM-HBV was observed in plated PTH [7], the slower kinetics of infection observed with HBV compared to WM-HBV in the tupaia-mouse model and the fact that the steady-state titres and cccDNA levels were similar, would suggest that other steps of the virus life-cycle may be involved. Chouteau et al. observed that WM-HBV was only

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able to infect cultivated primary human hepatocytes after being pseudo-typed with the L protein of HBV [23]. Whether the L-protein or any other cellular or viral factors contribute to the observed differences in efficiency of infection in tupaia chimeric mice needs further elucidation. However, sequence analysis failed to reveal the emergence of new viral populations in HBV infected mice, both before and after passage experiments, indicating that a strong selective pressure is not required to establish HBV infection in TPH repopulated mice. Further infection experiments are planned to investigate whether the evolutionary relatedness of the various HBV genotypes with respect to WM-HBV may affect the kinetics of infection in mice repopulated with PTH. Detection and determination of cccDNA in infected mice indicate that this model is suitable for studying cccDNA amplification and cccDNA half-life in vivo [18]. Though few animals were analyzed so far, our data indicate that after infection of mice either with HBV or WM-HBV, cccDNA and total intracellular viral DNA levels in engrafted mice were 10 times higher compared with human livers, though viral titres in serum were comparable [18]. This difference may be accounted for the lack of an immune system in uPA/ RAG-2 mice, which can suppress viral replication. Alternatively, it might be that viral secretion in tupaia cells is slightly impaired. However, the ratio of intracellular total DNA vs. cccDNA in grafted mice was within the range found in HBV-chronically infected human livers. Efficient repopulation of PTH was also achieved with cryopreserved cells, which greatly facilitates the use of this model. Due to the limited window available for transplantation in the uPA/RAG-2 mice, the ability to use cryopreserved tupaia liver cells offers the opportunity to perform transplantations at any time and without cell waste. Prompt responsiveness to antiviral drug treatment indicates that the tupaia/uPA/RAG-2 mice allow to directly investigate the effect of certain antiviral compounds on down regulation of viral replication, cccDNA stability and viral spread. Since infectious viral particles are produced for the life span of the animals, we plan to use the tupaia mouse model for long term antiviral studies, which is a major limitation of the antiviral drug evaluation performed in vitro using primary hepatocyte cultures.

Acknowledgements The authors thank M. Nassal for providing WM-HBV positive serum, C.S. Gibbs at Gilead Sciences for providing the adefovir dipivoxil, and Roswitha Reusch for excellent technical assistance. The work was supported by a grant from the Deutsche Forschungsgemeinschaft to J.P.(Pe 608/2-3/4). The Heinrich Pette Institute is supported by the German Federal Ministry of Health and the City of Hamburg.

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