Viral covalently closed circular DNA in a non-transgenic mouse model for chronic hepatitis B virus replication
Journal of Hepatology
Volume 44, Issue 2, Pages 267-274 (February 2006)
Tetsuo Takehara , Takahiro Suzuki , Kazuyoshi Ohkawa, Atsushi Hosui, Masahisa Jinushi, Takuya Miyagi, Tomohide Tatsumi, Yoshiyuki Kanazawa, Norio Hayashi
Background/Aims: The lack of small animal models supporting chronic hepatitis B virus (HBV) infection impedes the assessment of anti-viral drugs in the whole animal. Although transgenic mice have been used for this purpose, these models are clearly different from natural infection, because HBV is produced from the integrated HBV sequence harbored in all hepatocytes.
Methods: Balb/cA nude mice were hydrodynamically injected with a plasmid having 1.5-fold over-length of HBV DNA and analyzed for HBV replication.
Results: Hydrodynamically injected mice showed substantial levels of antigenemia and viremia for more than 1 year. Covalently closed circular DNA (cccDNA), the template of viral replication in natural infection, was produced in the livers and was critically involved in the long-term HBV production, because disruption of the pol gene of the inoculated DNA resulted in transient expression of HBV genes for less than 2 months. Administration of the IFNa gene transiently suppressed HBV DNA replication, but was not capable of eliminating HBV in this model.
Conclusions: In vivo gene transfer of a plasmid encoding HBV DNA can establish chronic viral replication in mice, which involves, at least in part, new synthesis of the HBV cccDNA episome, thus recapitulating a part of human HBV infection.
Hepatitis B virus (HBV) causes both transient and persistent infection in the human liver [1,2] When healthy adults are exposed to this virus, they usually develop acute transient infection with various degrees of liver injury, and, in most cases, have favorable outcomes. In contrast, when immunocompromised hosts such as newborn babies, drug abusers, and patients receiving immunosuppressive drugs, are infected with HBV, they cannot eliminate it and often suffer from chronic liver injury and hepatocellular carcinoma. Chronic carriage of this virus is a major health problem in many countries. Patients with chronic HBV infection are currently treated with interferon (IFN) or nucleotide analogs such as lamivudine and adefovir. However, the limited success and frequent recurrence after cessation of therapy require new strategies for terminating this viral infection.
Study of HBV replication in vivo is hampered by the lack of suitable small and well-characterized animal models; thus far, only chimpanzees and the tree shrew (Tupaia), a relatively uncharacterized animal, appear to support HBV infection . Several lines of transgenic mice have been established but HBV replication is generated from the integrated HBV sequence harbored in all hepatocytes, which is clearly different from the natural infection [4,5]. An alternative strategy is in vivo gene transfer of HBV DNA. Takahashi et al.  previously reported that intrahepatic injection of naked HBV DNA with cationic liposome can cross the species barrier and leads to HBV replication in rats. We and others have reported that hydrodynamics-based delivery of HBV DNA efficiently transduces murine livers and leads to HBV replication [7,8]. However, HBV replication in these models is terminated within a couple of weeks, presumably resulting from immunological elimination of HBV-expressing hepatocytes. Very recently, there have been reports of these models being applied for the assessment of anti-viral drugs [9-11]. However, the analysis may be hampered because this is a model of acute transient infection and would not allow observation of the long-term outcome.
In an attempt to develop a better long-term model, we hydrodynamically injected a plasmid encoding replication competent HBV DNA into immunocompromised mice and examined the kinetics of expression and replication of HBV. The mice produced HBV-related proteins for over 1 year, which appeared to be dependent on episomal HBV DNA replication in the liver, because the introduction of replication-incompetent HBV DNA led to transient expression of HBV genes. IFNa treatment of these mice showed transient repression of HBV replication but could not terminate it. These mice mimic a part of human HBV infection in terms of the template of viral replication and should be useful for analyzing the long-term outcome of anti-HBV therapy.
In the present study, we demonstrated that hydrodynamic injection of a plasmid encoding an overlength of HBV DNA into nude mice established long-term replication of HBV in the liver. Since hepatic damage was not observed, this model mimics the chronic carrier-like state of human HBV infections. This model reminds us of a 1988 report by Feitelson et al.,  in which they stated that intrahepatic injection of replication competent HBV DNA led to persistent HBs antigenemia as well as chronic liver injury in nude mice. They had no evidence of HBV replication such as production of Dane particles in the circulation. In a preliminary experiment, we intrahepatically injected pHBV1.5 into nude mice and monitored viral production in the serum. DNase I-resistant HBV DNA could not be detected in most mice tested; a small number of mice produced low levels of virus at 3 days after injection but not at later time points (our unpublished data). Thus, the transfection efficiency of hydrodynamic injection of HBV DNA appeared to be higher than that of intrahepatic injection. Despite the difference in liver damage observed among these studies, we considered the absence of hepatitis in the present model reasonable, since the T-cell immune response towards HBV-related antigens could not occur. Furthermore, it should be noted that the antigenemia as well as HBV production achieved by the hydrodynamic procedure was very reproducible, which is critically important when applying this model to evaluate the efficacy of anti-viral drugs.
The duration of hydrodynamics-based gene expression varies among reports from days to months [19,23,24]. The plasmid-based gene expression of our model terminated within 2 months, as demonstrated by the injection of replication-incompetent HBV DNA (mutant pHBV1.5). Replication-competent HBV DNA (wild-type pHBV1.5)-injected mice displayed a rapid decline of HBsAg production followed by relatively stable antigenemia for more than 1 year (Fig. 2B). Although the rapid decline observed in the first 2 weeks may reflect the plasmid-based gene expression, stable expression of HBsAg at later time points did not depend on residual plasmids in the livers, but required intracellular reproduction of HBV DNA. These results indicate that HBV replication in addition to immunological tolerance is critically important for long-term HBV expression in this system. Previous research on in vivo gene transfer  and transgenic mice  has indicated that HBV cccDNA, the template of HBV replication in natural infection, could not be detected in murine livers by Southern blot analysis. In the present study, we applied a highly sensitive PCR procedure and detected HBV cccDNA in pHBV1.5-injected livers. What is important is that the estimated numbers of HBV cccDNA per hepatocyte were 1 or 4, which should be sufficient for HBV gene expression. Taken together, the present study is the first demonstration of the production of viral cccDNA and its contribution to HBV replication in mice. Thus, the species restriction on the production of HBV cccDNA may not be as strict as has previously been believed.
Mutation of HBV DNA occurring during therapy with various nucleotide analogues leads to drug resistance and limits the success of these drugs for controlling HBV replication in humans [1,2]. Thus far, except for the in vitro recombinant HBV baculovirus system , there has been no useful model supporting reproduction of the HBV viral template as is the case of hepatitis C virus replicon systems [26,27]. Although HBV could not 'infect' murine hepatocytes, intracellular 'reinfection', namely recycling of HBV DNA occurs and leads to chronic viral production in the present model. Therefore, this model may provide a unique opportunity for analyzing possible mutations induced by long-term usage of various nucleotide analogs. Further study is needed to examine this possibility. Finally, intentional mutation could be easily introduced in inoculated DNA and a wide variety of mice with different genetic backgrounds can be used. The model presented here should enable analysis of viral as well as host factors that may regulate HBV replication.
3.1. Intravenous injection of pHBV1.5 leads to hepatitis B antigenemia as well as hepatic expression of HBcAg for more than 1 year
We injected 25μg of pHBV1.5, which contains 1.5-fold overlength HBV DNA, into the tail veins of nude mice with acute circulatory overload. To investigate the expression of HBV, the presence of HBV transcripts was analyzed by Northern blot in various organs from mice sacrificed at 3 days after the injection (Fig. 2A). Two major bands corresponding to 3.5 and 2.4/2.1kb transcripts were detected in the liver but not in other tissues including the kidney, spleen, thymus, lung, heart, and brain. The levels of HBsAg and HBeAg in the serum were serially determined by a quantitative CLIA method (Fig. 2B). Although the levels of HBsAg rapidly decreased 1.5log within the first 2 weeks, all mice were persistently positive for HBsAg and HBeAg for more than 1 year. Immunohistochemical analysis revealed that around 4% of the hepatocytes were positive for HBc at 3 days after injection (Fig. 2C). HBcAg-positive cells gradually decreased in number but were still detected at one year after the injection (Fig. 2D). Although data are not shown, hepatic damage could not be detected, as evidenced by biochemical and histological analysis, throughout the course, except during the first week; it resulted from hemorrhagic destruction of the liver due to hydrodynamic pressure. Taken together, these results indicated that hydrodynamics-based delivery of a plasmid encoding replication-competent HBV DNA can establish specific expression of HBV genes in the liver and persistent expression without significant liver injury for a period of more than 1 year.
3.2. Long-term productive replication of HBV DNA
To examine if viral particles are produced into the circulation, sera obtained at 3 days after pHBV1.5 injection was treated with DNase I and fractionated by sucrose density gradient centrifugation. As shown in Fig. 3A, when each fraction was assayed in PCR for the presence of HBV DNA, the strongest signal was observed in the fraction with a density of 1.21g/ml, corresponding to the density of HBV particles derived from human sera . In addition, when serum was pre-treated with detergent before the centrifugation, the positive fraction shifted to a density of 1.28g/ml, suggesting that detergent treatment releases core particles from HBV particles by removing the envelope.
To examine the kinetics of viremia, we examined the levels of DNase I-resistant HBV DNA in serum by real-time PCR analysis (Fig. 3B). The levels of HBV DNA were as high as 1X107copies/ml at 3 days after the injection and gradually decreased by 1.5log over 1 year.
3.3. Long-term expression of HBV is dependent on HBV replication
The extremely long-term expression and carriage of HBV in this system led us to examine whether episomal replication could affect the kinetics of expression of HBV-related genes. Toward this goal, we introduced point mutation in the pol gene of pHBV1.5 which could produce the truncated form of the HBV polymerase without affecting the expression of any other HBV-related proteins. Mice hydrodynamically injected with mutant pHBV1.5 produced HBsAg as well as HBcAg at levels similar to those of wild-type pHBV1.5-injected mice 3 days after injection (Fig. 4A and B). However, mutant pHBV1.5-induced expression of HBsAg, HBeAg and HBcAg was terminated within 2 months, in striking contrast to wild-type pHBV1.5-induced gene expression (Fig. 1B and D). Northern blot analysis confirmed the transient expression of HBV genes after injection of mutant pHBV1.5 (Fig. 4C).
HBV DNA polymerase binds to the 5' end of its own mRNA template, and the complex is then packaged into nucleocapsids, where viral DNA synthesis occurs . HBV genomic DNA produced via the reverse transcription pathway predominantly consists of relaxed-circular DNA with a complete minus strand and a partially synthesized plus strand. In natural HBV infection in humans, part of the nucleocapsids migrates to the nucleus where relaxed-circular DNA is converted to cccDNA that serves as a template for transcription . The finding in the present model of long-term expression of HBV involving HBV DNA replication suggested that viral cccDNA may be produced in murine livers and work as a transcriptional template for HBV expression, in addition to the inoculated plasmid. To examine the presence of cccDNA in the liver, we used a PCR procedure which selectively detects cccDNA (Fig. 1). We also checked for the presence of inoculated plasmids by amplifying the ampicillin resistance gene by PCR. The authenticity of the cccDNA detection was confirmed by the detection of a specific signal from liver tissues of patients with chronic hepatitis B, but not from the serum of patients or pHBV1.5 (Fig. 5A). Viral cccDNA was clearly detected in wild-type pHBV1.5-injected livers at 3 days as well as 3 months after the injection (Fig. 5B). As expected, cccDNA was not detected in mutant pHBV1.5-injected livers. The levels of cccDNA were measured by real-time PCR (n=5 for each time point) and results were 2.4X107 and 6.0X105 copies per gram of liver tissue at 3 days and 2 months after the injection, respectively. Since the liver approximately contains 1.1X108 of hepatocytes, the average copy numbers of HBV cccDNA per core Ag-positive hepatocyte could be estimated to be 1 or 4. Ampicillin resistance gene was similarly amplified from both wild-type pHBV1.5- and mutant pHBV1.5-injected livers. The fact that HBV gene expression was terminated within 2 months upon injection of mutant pHBV1.5 clearly indicates that the presence of residual plasmids in the livers at later time points is not sufficient for the expression of detectable levels of HBV genes; this is consistent with a previous report  demonstrating that transgene expression is rapidly terminated after hydrodynamic gene delivery despite the persistence of plasmid DNA in the livers. These results support the idea that viral cccDNA is critically involved in the long-term expression and carriage of HBV in this model.
3.4. Administration of IFNa gene transiently suppressed HBV DNA replication and failed to eradicate viral template
We next sought to examine the potential usefulness of this model for the assessment of anti-viral drugs. To examine the effect of IFNa in the phase of cccDNA-dependent HBV replication, we injected either pCMV-IFNa1 or pCMV at 70 days after pHBV1.5 injection. Injection of pCMV-IFNa1 led to substantial IFNa production at day 1 (Fig. 6A), although IFNa could not be detected in the mock-injected mice (data not shown). The levels of IFNa after pCMV-IFNa1 injection rapidly declined at day 3 and could not be detected at day 28. Injection of pCMV-IFNa1 significantly suppressed viral production at day 3 but did not affect HBs production (Fig. 6B and C); this is consistent with previous findings [15,21] that IFNa suppressed HBV replication at a step of reverse transcription. In spite of the substantial suppression of HBV production at day 3, the levels of viral titers of mice injected with pCMV-IFNa1 increased to levels similar to those of pCMV-injected mice at day 14 and later. These results indicate that IFN treatment substantially suppressed viral replication, but could not eliminate the viral template from the infected host. This model should be useful for assessing anti-viral therapy aimed at eradication of the viral template.
2. Materials and methods
2.1. Plasmids and mutagenesis
Plasmid pHBV1.5 containing an overlength (1.5-mer) copy of HBV DNA (GenBank accession no. AF305422) has been described previously . A plasmid containing mutant HBV DNA carrying a stop codon instead of 54Trp of the pol gene was generated from pHBV1.5 by a GeneTailor Site-Directed Mutagenesis system (Invitrogen, Carlsbad, CA) and verified by sequencing. The site of the mutation was designed not to affect the expression of any HBV-related genes except for the pol gene. A plasmid coding the murine IFNa1 gene, pCMV-IFNa1, was generously provided by Dr Daniel J.J. Carr (University of Okulahoma, Health Science Center) .
Specific pathogen-free female Balb/cA nude mice were purchased from Clea Japan, Inc. (Tokyo, Japan) and were used at the age of 5 to 6 weeks. They were housed under conditions of controlled temperature and light with free access to food and water at the Institute of Experimental Animal Science, Osaka University Graduate School of Medicine. All animals received humane care and study protocol complied with the institution's guideline.
2.3. Injection of naked plasmid DNA
Plasmid DNA was prepared using an EndoFree plasmid system (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Hydrodynamic injection of plasmid DNA was performed according to previous reports [13,14]. In brief, 25μg of plasmid DNA was diluted with 2.0ml of lactated Ringer's solution and injected into the tail vein using a syringe with a 30-gauge needle. DNA injection was completed within 8 to 15s.
2.4. Nothern blot
Total tissue RNA was isolated with Isogen (Nippon Gene, Toyama, Japan), and then 30μg of total RNA was analyzed by Northern blotting with the HBV adw2 probe, as described previously .
For immunohistochemical detection of HBc protein, tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. After being deparaffinized, sections 4μm thick were incubated with anti-HBc antibody (Dako, Denmark), followed by immunoperoxidase staining using the ABC procedure (Vector Laboratories, Burlingame, CA) and counterstaining with hematoxylin.
2.6. Detection of hepatitis B antigens in serum
Under light anesthesia using sevoflurane, animals were bled from the retro-orbital vessels. Serum HBs antigen and HBe antigen were measured by chemiluminescent immunoassay (CLIA system, Abbott Laboratories, North Chicago, IL).
2.7. Real-time detection of HBV DNA in serum
Serum was treated with DNase I (Takara, Tokyo, Japan) and then proteinase K. DNA was extracted from the sera by a QIAamp DNA blood isolation system (Qiagen). HBV DNA was quantified by using real-time polymerase chain reaction (PCR) technology (Applied Biosystems, Foster City, CA) as described previously . Primers and fluorescent probes are as follows: sense (nucleotides 168-188), 5'-CACATCAGGATTCCTAGGACC-3'; antisense (nucleotides 341-321), 5'-GGTGAGTGATTGGAGGTTGG-3'; probe (nucleotides 244-269), 5'-FAM-CAGAGTCTAGACTCGTGGTGGACTTC-3'.
2.8. Density analysis of HBV particles in serum
DNase I-treated serum was clarified by centrifugation at 15,000rpm for 15min using a 0.45μm membrane filter. The clarified serum was layered on top of a 10-60% discontinuous sucrose gradient. Centrifugation was carried out at 141,000g for 48h. Fractions were collected from the bottom of the tube. After treatment with proteinase K, DNA was isolated from each fraction and applied for analysis of HBV DNA by PCR . In an additional experiment, DNase I-treated serum was incubated with 1% Nonidet P-40 and 0.3% 2-mercaptoethanol for 16h at 37 C, and then used for density analysis.
2.9. Detection of HBV covalently closed circular DNA (cccDNA)
DNA was isolated from liver tissues by using a DNeasy Tissue kit (Qiagen). PCR detection of cccDNA was performed according to the procedure of Jun-Bin et al.  with some modification (Fig. 1). The PCR product was analyzed on a 1.2% agarose gel by electrophoresis. In some experiments, cccDNA was quantified using real-time PCR. To calculate the number of cccDNA per HBcAg-positeve hepatocyte, the total number of hepatocytes was estimated from the genomic DNA content in the murine liver under the assumption that the liver is about 70% hepatocytes. In addition, ampicillin resistance gene in the plasmids was amplified by using a sense primer (5'-TATGGCTTCATTCAGCTCCG-3') and an antisense primer (5'-TCGAACTGGATCTCAACAGC-3').