The use of the hydrodynamic HBV animal model to study HBV biology and anti-viral therapy.

A simple reproducible and versatile small animal model for hepatitis B virus (HBV) infection is still unavailable. We have generated a simple transient liver-targeted transgenic mouse. Hydrodynamics tail vein injection of a head-to-tail dimer of adw HBV genome (pHBVadwHTD) into immunocompetent mice generated HBsAg and HBeAg expression in both serum and hepatocytes, followed by seroconversion. The injection of pHBVadwHTD into SCID mice generated prolonged HBsAg and HBeAg antigenemia and HBV viremia. Our results demonstrate that hydrodynamic injection of naked DNA could support the generation of HBV particles. We used this model for the assessment of anti-viral agents. Administration of our human monoclonal antibodies, HBV-Ab17(XTL) and HBV-Ab19(XTL), as well as Lamivudine (3TC) treatment suppressed HBV viremia. The model presented herein supports long and stable expression of HBV and will enable determination of various biological questions related to HBV life cycle, mutants and could enhance the development of anti-viral reagents.     

Viral covalently closed circular DNA in a non-transgenic mouse model for chronic hepatitis B virus replication

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 IFNalpha 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.     

Acute hepatitis in rats expressing human hepatitis B virus transgenes.

The molecular mechanisms responsible for hepatocyte death and the events leading to viral clearance in hepatitis B virus (HBV) infections are not well understood. Elucidation of the mechanisms involved have been complicated by the difficulty of infecting human hepatocytes with HBV in vitro and the lack of an appropriate animal model. We report an animal model of human HBV infection by in vivo transfection. We have directly introduced a replication-competent, cloned HBV construct into rat liver by using a membrane fusion-promoting cationic lipid. HBV mRNA and 3.2-kb HBV DNA were expressed in the liver by this in vivo transfection method. In the majority of rats, HBV virions and hepatitis B e antigen were found in the blood 3-7 days after transfection, after which antibody to the e antigen appeared. Two to three weeks after the transfection, glutamic-pyruvic transaminase levels were elevated in serum, hepatocyte death and lymphocyte infiltration were observed in the vicinity of the portal vein of liver, and HBV virions were no longer detected in the serum. Thus, transfection of HBV into rats resulted in histological and serological changes comparable to HBV-induced acute hepatitis in humans. In contrast, no hepatocellular injury was observed in T-lymphocyte-deficient nude rats transfected with the same HBV construct, and viremia was substantially prolonged, providing direct evidence that T lymphocytes play an essential role in liver cell injury and in the clearance of HBV. This rat hepatitis model will be useful for studying pathogenesis of HBV infection.     

Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation

No specific drugs are currently available against hepatitis delta virus (HDV), a defective virus leading to the most severe form of chronic viral hepatitis in man. The lack of convenient HDV infection models has hampered the development of effective therapeutics. In this study, na?ve and hepatitis B virus (HBV) chronically infected humanized uPA/SCID mice were employed to establish a small animal model of HBV/HDV coinfection and superinfection. For preclinical antiviral drug evaluation, the GMP version of the myristoylated preS-peptide (Myrcludex-B), a lipopeptide derived from the pre-S1 domain of the HBV envelope, was applied to prevent de novo HBV/HDV coinfection in vivo. Virological parameters were determined at serological and intrahepatic level both by real-time polymerase chain reaction (PCR) and by immunohistochemistry. Establishment of HDV infection was highly efficient in both HBV-infected and na?ve chimeric mice with HDV titers rising up to 1 × 10E9 copies/mL. Notably, HDV superinfection led to a median 0.6log reduction of HBV viremia, which although not statistically significant suggests that HDV may hinder HBV replication. In the setting of HBV/HDV simultaneous infection, a majority of human hepatocytes stained HDAg-positive long before HBV spreading was completed, confirming that HDV can replicate intrahepatically also in the absence of HBV infection. Furthermore, the increase of HBV viremia and intrahepatic cccDNA loads was significantly slower than in HBV mono-infected mice. Treatment with the HBV entry inhibitor Myrcludex-B, efficiently hindered the establishment of HDV infection in vivo. CONCLUSION: We established an efficient model of HBV/HDV infection to exploit mechanisms of viral interference in human hepatocytes and to test the efficacy of an HDV-entry inhibitor in vivo.     

骨髓间充质干细胞对乙型肝炎病毒的易感性及与无涎糖蛋白受体的关系

目的 评价骨髓间充质干细胞(BMSC)向肝细胞诱导过程中对HBV的易感性及无涎糖蛋白受体(ASGPR)对BMSC感染HBV的作用.方法 体外使用肝细胞生长因子、成纤维细胞生长因子-4和表皮生长因子,将BMSC诱导分化为肝细胞.检测乙型肝炎患者BMSC的HBV感染情况,并对原代及诱导培养后的BMSC进行体外HBV感染实验,检测BMSC感染后的HBsAg、HBcAg表达情况,并检测BMSC诱导前后ASGPR的表达.每个实验采用来自不同的5个标本,分别重复3次,数据统计采用非参数检验.结果 诱导培养第6天开始,BMSC开始表达甲胎蛋白(AFP)、细胞角蛋白18(CK18)和Alb,并随着诱导时间延长,CK18及Alb表达逐渐增多,而AFP则逐渐减少,并具有糖原合成、尿素分泌及Alb合成的肝细胞功能.BMSC在体内及体外都不能被HBV感染,经过向肝细胞诱导之后,仍然不能被感染,ASGPR在BMSC向肝细胞诱导后表达增多,但是与对照组HepG2细胞相比,仍然呈低水平表达.结论 BMSC在体内外能抵抗HBV感染.ASGPR可能是导致HBV不能感染BMSC的重要原因之一.     

What risk is a health care worker infected with hepatitis B or C virus for his patients?

Presently, there are no legislative standards in the Czech Republic banning health care workers with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection to do activities with a risk of the transmission of these viral infections to patients (surgeries and other invasive interventions). In a range of developed countries in the world individuals with chronic HBV infection, HBeAg positive individuals, have a restricted access to the risk interventions. A quantitative assessment of viremia is important in the health care workers infected with HBeAg-minus mutant of the virus. There are particular critical viremia values set up (serum HBV DNA levels) which exceeding in the health care workers leads to banning them to do the risk interventions. In cases of proved transmission of hepatitis B infection from a health care worker to a patient, the ban of doing risk interventions is a rule. Transmission of HCV infection from a health care worker to a patient is much less probable so the individuals with chronic hepatitis C are usually not forbidden to make invasive procedures. An exception are cases when there was a patient infected by a particular health care worker in the past. There are various attitudes to the health care workers with chronic HBV or HCV infection in various countries of the world. A necessity to reach a definite consensus is necessary. The first step to it are common recommendations of 12 European countries and the USA which are repeatedly cited in the text. We can expect that these problems will have to be solved very soon in the Czech Republic too.     

Effect of Combined Treatment of Serum Containing Hepatitis B Virus with Beta-Propiolactone and Ultraviolet Irradiation

The effect of cold sterilization by β-propiolactone (β-PL) and ultraviolet (UV) irradiation of serum contaminated with infectious hepatitis B virus (HBV) was investigated in chimpanzees. Chimpanzees given 0.1 ml/kg of the undiluted HBV serum estimated to contain 10⁷-10⁸ CID₅₀/ml developed acute hepatitis B infection 4 weeks after inoculation. Chimpanzees injected with the same undiluted hepatitis serum treated with β-PLAJV developed hepatitis B infection 14 weeks later. Based on the published linear relationship between log dose of HBV and incubation period in chimpanzees this indicates a 10⁶-fold reduction in infectivity titer. Animals inoculated with the serum diluted 1:1,000 showed manifest hepatitis B infection 11 weeks later. Animals inoculated with serum diluted 1:1,000 and then cold sterilized with -PL/U V showed no signs of hepatitis B infection. Sensitive proteins are not denaturated by β-PL/UV cold sterilization.     

Rapid emergence of telaprevir resistant hepatitis C virus strain from wildtype clone in vivo

Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3-4A protease. However, the emergence of drug-resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra-deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low-level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT-9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower-level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir-resistant NS3 V36A variants emerged 2 weeks after the start of treatment. Conclusion: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir-resistant HCV was induced by mutation from the wildtype strain of HCV in vivo.     

A novel cDNA-uPA/SCID/Rag2−/−/Jak3−/− mouse model for hepatitis virus infection and reconstruction of human immune system

Although human hepatocyte-transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA-urokinase-type plasminogen activator (uPA)/SCID/Rag2^−/−/Jak3^−/− mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)-A24 resulted in establishment of a highly replaced liver in cDNA-uPA/SCID/Rag2^−/−/Jak3^−/− mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti-HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA-A24 donor resulted in establishment of 22.6%–81.3% human CD45-positive mononuclear cell chimerism in liver-infiltrating cells without causing graft-versus-host disease in cDNA-uPA/SCID/Rag2^−/−/Jak3^−/− mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte-specific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2^−/−/Jak3^−/− mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection.     

Selective transmission of hepatitis B virus after percutaneous exposure

In 3 clusters of postsurgical hepatitis B virus (HBV) infection, HBV DNA sequence mismatches were observed between the transmitting surgeons and the patients whom they infected. Sequence analysis of clones amplified from the C gene of HBV suggested that the mismatches were due to transmission of a minority variant in the circulation of each surgeon. Compared with 5 other transmitters from whom transmission of the dominant variant was demonstrated, the 3 surgeons who transmitted minority variants carried significantly more heterogeneous HBV populations. Transmission of minority variants was not correlated with the transmitters' hepatitis B antigen status, the presence of the position 1896 precore mutant, or the level of HBV viremia. In 1 cluster, a variant comprising <10% of the HBV population circulating in the transmitting surgeon established infection in all 3 patients who acquired HBV through him, which substantiates the phenomenon of true selection.     

Secreted virus-encoded proteins reflect murine cytomegalovirus productivity in organs.

Mouse infection with murine cytomegalovirus (MCMV) is an established model for studying human cytomegalovirus infection. In this study, the relationship was analyzed between MCMV activity in organs of infected mice and the presence of infectious virus (viremia), viral genomes (DNAemia), or secreted virus-encoded proteins in the blood. For the latter, 2 recombinant viruses were constructed that encode for the hepatitis B virus surface antigen and the secreted alkaline phosphatase, respectively, as secreted marker proteins. The secreted markers correlated better with the infection in organs than DNAemia and viremia. The marker protein assays can serve as practical and sensitive tools for longitudinal monitoring of MCMV infection in individual mice.     

Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia

Analysis of hepatitis B virus (HBV) kinetics with mathematical models may disclose new aspects of HBV infection and host response mechanisms. To determine the kinetics of virion decay from the blood of patients in different phases of chronic infection, we applied mathematical modeling to real-time polymerase chain reaction assays, which enable quantification of viremia and intrahepatic HBV productivity by measuring both copy number and activity of covalently closed circular DNA (relaxed circular DNA/covalently closed circular DNA) in the liver of 80 untreated chronically active HBV carriers (38 hepatitis B e antigen [HBeAg]-positive and 42 HBeAg-negative individuals). We found that the half-life of circulating virions is very fast (median 46 and 2.5 minutes in HBeAg-positive and HBeAg-negative individuals, respectively) and strongly related to viremia, with clearance rates significantly accelerating as viral loads decrease. To investigate whether immune components can influence the kinetics of virion decay, we analyzed viral dynamics in immunodeficient urokinase-type plasminogen activator chimera mice. Virion half-life in mice (range, 44 minutes to >4 hours) was comparable to estimates determined in high viremic carriers, implying that clearance rates in these patients are mostly determined by common nonspecific mechanisms. Notably, the lack of correlation between virion half-life and viremia in mice indicated that immune components significantly accelerate virion clearance rates in individuals with low titers. CONCLUSION: Our analyses suggest that both host defense mechanisms and levels of circulating virions affect the kinetics of HBV decay assessed in the serum of chronic carriers. Identification of the factors affecting clearance rates will be important for future antiviral drug developments and it may give insights into the mechanisms involved in clearance of other chronic infections, such as human immunodeficiency virus and hepatitis C virus.     

SCID-hu小鼠在HBV感染研究中的应用

乙型肝炎病毒由于其特殊的嗜肝性,不易在其他动物模型重现其感染过程．SCID小鼠作为一种先天性T,B细胞双重缺陷动物,为异种移植研究提供了极大的帮助．随着SCID-hu模型的出现,人们通过移植人肝细胞、人外周血淋巴细胞等各种方法建立人鼠嵌合模型,让其感染乙型肝炎病毒,来研究乙型肝炎的发病机制．嵌合模型模拟了疾病在人体内的过程,据此所得的研究结果具有更强的说服力．     

Expression and replication of hepatitis B virus genome in transgenic mice.

We produced transgenic mice by microinjecting a partial tandem duplication of the complete hepatitis B virus (HBV) genome into fertilized eggs of C57BL/6 mice. One of eight transgenic mice was a high producer for HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. The HBV genomes were transmitted to the next generation and these F1 mice also produced HBsAg and HBeAg. mRNAs of 3.5, 2.1, and 0.8 kilobases were detected in the livers and the kidneys of these mice. In addition, a 0.8-kilobase RNA was detected in the testis. Single-stranded and partially double-stranded HBV DNAs were shown to be produced in the cytoplasm of the liver and kidneys. These HBV DNAs were associated with the core particles, indistinguishable from nucleocapsid produced in an infected human liver. Viral genome DNA was detected in the serum. These results demonstrate that the HBV genome integrated into the mouse chromosome acted as a template for viral gene expression, allowing viral replication. Thus, these transgenic mice should be useful for detailed studies of the replication and expression of HBV and for pathological studies of hepatitis, including the development of hepatocellular carcinoma.     

A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice

Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transferring the Simian Virus 40 Large T-Antigen into primary human hepatocytes. These cells were stably transfected with a full-length HBV genome to generate a clone that expresses HBV genes and replicates HBV. The HBV-producing cells were transplanted into the livers of mice with combined immunodeficiency (Rag-2 deficient) by intrasplenic injection. Survival of the engrafted human hepatocytes was shown in several ways: fluorescent in situ hybridization (FISH) with a human-chromosome-specific DNA probe (human alpha satellite), dot-blot hybridization of the genomic DNA extracted from liver biopsy specimens with a human-specific Alu repetitive DNA probe, Blur-8, as well as with an HBV DNA probe, and secretion of human proteins into plasma. Histological examination of mouse liver up to 8 months following human cell transplant shows completely normal architecture. Determination of plasma HBV DNA levels indicated that engrafted cells secreted 3x10(7) to 3x10(8) virions per mL into the blood, and HBsAg was detected in plasma. This new murine model of HBV viremia should be useful for in vivo HBV studies.     

Outcome of hepatitis B virus infection in homosexual men and its relation to prior human immunodeficiency virus infection.

To investigate the effect of human immunodeficiency virus type 1 (HIV-1) infection on subsequent hepatitis B virus (HBV) infection, HIV antibody was sought in homosexual men who developed HBV infection during a hepatitis B vaccine trial. Among 134 unvaccinated HIV-1-negative men, 7% became HBV carriers, 64% had viremia, and 42% had clinical illness. Among vaccinated HIV-1-negative men, HBV infection severity decreased with number of vaccine doses administered. When adjusted for prior hepatitis B vaccination status, persons with HIV-1 infection preceding HBV infection had a significantly higher risk of developing HBV carriage, viremia, prolonged ALT elevation, and clinical illness. Among HIV-1-infected men, the risk of HBV carriage was increased in unvaccinated persons (21%) and those who failed to respond to vaccination (31%) and further increased in those who received vaccine doses at the time they developed new HBV infection (56%-80%), suggesting inactivated hepatitis B vaccine may temporarily impair the immune response to HBV infection in HIV-1-infected persons. HIV-1 infection was also associated with reduced alanine aminotransferase elevations during the first 36 months of follow-up of men who became HBV carriers.     

Reappearance of hepatitis D virus (HDV) replication in chronic hepatitis B virus carrier chimpanzees rechallenged with HDV

Four chronic hepatitis B virus (HBV) carrier chimpanzees, which had apparently cleared hepatitis D virus (HDV) after a first experimental challenge with HDV, were reinoculated with a homologous strain of HDV. All animals had reappearance of low levels of serum HDV RNA and transient, mild alanine aminotransferase (ALT) elevations, which in two cases correlated with HDV RNA positivity. Plasmas obtained from two chimpanzees after rechallenge were inoculated into two other chronic HBV carrier animals that had recovered from a previous HDV infection. A similar reappearance of HDV RNA in serum (without ALT elevation) was noticed. These same plasmas, however, when inoculated into a chronic HBV carrier chimpanzee never exposed to HDV caused a severe acute hepatitis D. Rechallenge with HDV in chimpanzees apparently recovered from a first HDV infection resulted in the reappearance of HDV replication, sometimes associated with hepatitis. This can be interpreted as reinfection with HDV, but other explanations are possible. Although the serum level of HDV RNA observed after rechallenge with HDV is low, its transmission to individuals susceptible to HDV infection can result in severe acute hepatitis D.     

Successful combination therapy of polyarteritis nodosa associated with a pre-core promoter mutant hepatitis B virus infection.

BACKGROUND/AIMS: To describe the clinical and virological evolution of a polyarteritis nodosa (PAN) case associated with a hepatitis B virus (HBV) pre-core promoter mutant infection that was successfully treated with plasma exchanges, corticosteroids, and interferon alpha (IFN-alpha). METHODS: Viral markers were used, including HBV DNA quantified by the branched DNA assay and detected by PCR, the HBV genome sequence, pre-S1Ag and anti-HBC IgM which were studied throughout the treatment period and the entire follow-up in the serum, while the presence of virus in extrahepatic sites was detected by immuno-staining. RESULTS: The patient was infected with a typical pre-core promoter mutant harboring four point mutations. Pre-S1Ag was cleared rapidly from serum, most likely via the formation of immune complexes since HBV DNA declined more progressively. Viral infection was then cleared after a second episode of hepatocyte lysis. This was accompanied by a recovery from all clinical manifestations. CONCLUSIONS: The favorable treatment outcome observed in this first case of pre-core promoter HBV mutant associated PAN underlines that combination therapy based on IFN-alpha can clear pre-core promoter HBV infection and cure PAN. It also provides new insight in the pathogenesis of HBV associated PAN.