HBV转基因小鼠--乙型肝炎研究的重要工具

HBV感染具有严格的种属特异性和组织亲嗜性,所以可用于研究的动物模型很少。随着转基因技术的出现和发展,人们通过研制转基因动物模型来研究病毒致病机理。把HBV的DNA或其片段转入小鼠受精卵建立起来的HBV转基因小鼠,已被广泛地应用于乙型肝炎的研究中,主要体现在以下3个方面：研究HBV的致病机制、与肝细胞癌的关系及寻找、评价治疗乙肝的方法。     

Hepatitis viruses and hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide. Hepatitis viruses, such as the hepatitis B virus (HBV) and hepatitis C virus (HCV) are undoubtedly listed in the etiology of HCC. Studies show that, in the near future, viral hepatitis will carry increasing weight in the etiology of HCC. This review briefly discusses the known carcinogenic effects of HBV and HCV in the light of experimental and human studies. The data show that viral proteins may directly interfere with gene products responsible for cell proliferation and cell growth. Many other signal transduction cascades may be affected as well. Direct integration of HBV viral sequences into the host genome increases the genomic instability. The genomic imbalance allows the development and survival of malignant clones bearing defected genomic information. HBV and HCV infection induces indirect and direct mechanisms through cellular damage, increased regeneration and cell proliferation, therefore enhancing the development of HCC.     

慢性肝炎中乙型和丙型病毒混合感染的重症化研究

在慢性肝炎中,乙、丙型病毒性肝炎混合感染相当多见,可使肝炎慢性化、重症化,肝组织损伤加重,肝硬化(LC)和肝癌(HCC)发生率增加[1]。本文应用血清学和分子生物学方法对196例肝病患者的血清进行检测,初步探讨了乙型肝炎病毒(Hepatitis B virus,HBV)、丙型肝类病毒(Hepatitis C virus,HCV)的复制状况以及两者间的相互作用与预后的关系。1材料和方法1.1病例受检的196例病例均为2004年1月至2005年7月我院住院及门诊病人,男149例,女47例,年龄15~82岁,其中慢性肝炎(CH)患者139例,肝硬化(LC)患者42例,肝癌(HCC)患者15例。所有病例诊断符合…     

Human oncogenic viruses: Hepatitis B and hepatitis C viruses and their role in hepatocarcinogenesis

Chronic infections caused by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) are the main risk factors for the development of hepatocellular carcinoma (HCC) in humans. Both viruses cause a wide spectrum of clinical manifestations ranging from healthy carrier state to acute and chronic hepatitis, liver cirrhosis, and HCC. HBV and HCV belong to different viral families (Hepadnoviridae and Flaviviridae, respectively); they are characterized by different genetic structures. Clinical manifestations of these viral infections result from the interaction between these viruses and host hepatocytes (i.e. between viral and cell genomes). Proteins encoded by both viruses play an important role in processes responsible for immortalization and transformation of these cells. Chronic inflammation determined by host immune response to the viral infection, hepatocyte death and their compensatory proliferation, as well as modulation of expression of some regulatory proteins of the cell (growth factors, cytokines, etc.) are the processes that play the major role in liver cancer induced by HBV and HCV.     

Enhanced host immune responses in presence of HCV facilitate HBV clearance in coinfection

Hepatitis B virus (HBV)/Hepatitis C virus (HCV) coinfection is frequently observed because of the common infection routine. Despite the reciprocal inhibition exerted by HBV and HCV genomes, the coinfection of HBV and HCV is associated with more severe forms of liver diseases. However, the complexity of viral interference and underlying pathological mechanism is still unclarified. With the demonstration of absence of direct viral interplay, some in vitro studies suggest the indirect effects of viral-host interaction on viral dominance outcome. Here, we comprehensively investigated the viral replication and host immune responses which might mediate the interference between viruses in HBV/HCV coinfected Huh7-NTCP cells and immunocompetent HCV human receptors transgenic ICR mice. We found that presence of HCV significantly inhibited HBV replication in vitro and in vivo irrespective of the coinfection order, while HBV did not affect HCV replication. Pathological alteration was coincidently reproduced in coinfected mice. In addition to the participation of innate immune response, an involvement of HCV in up-regulating HBV-specific immune responses was described to facilitate HBV clearance. Our systems partially recapitulate HBV/HCV coinfection and unveil the uncharacterized adaptive anti-viral immune responses during coinfection, which renews the knowledge on the nature of indirect viral interaction during HBV/HCV coinfection.     

Chimeric Mouse Model for the Infection of Hepatitis B and C Viruses

While the chimpanzee remains the only animal that closely models human hepatitis C virus (HCV) infection, transgenic and immunodeficient mice in which human liver can be engrafted serve as a partial solution to the need for a small animal model for HCV infection. The established system that was based on mice carrying a transgene for urokinase-type plasminogen activator (uPA) gene under the control of the human albumin promoter has proved to be useful for studies of virus infectivity and for testing antiviral drug agents. However, the current Alb-uPA transgenic model with a humanized liver has practical limitations due to the inability to maintain non-engrafted mice as dizygotes for the transgene, poor engraftment of hemizygotes, high neonatal and experimental death rates of dizygous mice and a very short time window for hepatocyte engraftment. To improve the model, we crossed transgenic mice carrying the uPA gene driven by the major urinary protein promoter onto a SCID/Beige background (MUP-uPA SCID/Bg). These transgenic mice are healthy relative to Alb-uPA mice and provide a long window from about age 4 to 12 months for engraftment with human hepatocytes and infection with hepatitis C or hepatitis B (HBV) viruses. We have demonstrated engraftment of human hepatocytes by immunohistochemistry staining for human albumin (30-80% engraftment) and observed a correlation between the number of human hepatocytes inoculated and the level of the concentration of human albumin in the serum. We have shown that these mice support the replication of both HBV and all six major HCV genotypes. Using HBV and HCV inocula that had been previously tittered in chimpanzees, we showed that the mice had approximately the same sensitivity for infection as chimpanzees. These mice should be useful for isolating non-cell culture adapted viruses as well as testing of antiviral drugs, antibody neutralization studies and examination of phenotypic changes in viral mutants.     

丙型肝炎病毒结构基因转基因小鼠的建立

为探讨丙型肝炎病毒（ＨＣＶ）结构基因在ＨＣＶ感染中的致病性,构建了中国丙型肝炎病毒５ＵＴＲ区与结构基因区（Ｃ＋Ｅ１＋Ｅ２）的表达质粒,并通过显微注射法将其接种入小鼠受精卵内制备转基因小鼠。共注射受精卵４１０枚,存活３１２枚,植入后产仔６０只;转基因鼠尾部组织ＰＣＲ法ＤＮＡ检测证明有靶基因的整合;转基因小鼠的肝、肾、脾、心、肺、小肠、血中均有靶基因的转录,而在脑组织中无转录。３只Ｇｏ代整合小鼠经与正     

Molecular aspects of hepatitis B viral infection and the viral carcinogenesis

Of many viral causes of human cancer, few are of greater global importance than the hepatitis B virus (HBV). Over 250 million people worldwide are persistently infected with HBV. A significant minority of these develop severe pathologic consequences, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Earlier epidemiological evidence suggested a link between chronic HBV infection and HCC. Further, the existence of related animal viruses that induce acute and chronic infections of the liver, and eventually HCC, confirms the concept that HBV belongs to one of the few human oncogenic viruses. Although it is clear that chronic HBV infections are major risk factors, relatively little is understood about how the viral factors contribute to hepatocarcinogenesis. This review will introduce molecular aspects of the viral infection, and highlight recent findings on the viral contribution to hepatocarcinogenesis.     

Hepatitis C virus core protein: intriguing properties and functional relevance

Hepatitis C virus (HCV) often causes a prolonged and persistent infection, and an association between hepatocellular carcinoma (HCC) and HCV infection has been noted. The pathogenesis of liver damage is at least in part related to virus-mediated factors. Understanding the molecular basis of pathogenesis is a major challenge in gaining insight into HCV-associated disease progression. Recent experimental evidence using HCV cloned genomic regions suggests that the core protein has numerous functional activities. These include its likely role in encapsidation of viral RNA, a regulatory effect on cellular and unrelated viral promoters, interactions with a number of cellular proteins, an modulatory role in programmed cell death or apoptosis under certain conditions, involvement in cell growth promotion and immortalization, induction of HCC in transgenic mice, and a possible immunoregulatory role. These intriguing properties suggest that the core protein, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection.     

Susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species

The susceptibility of laboratory mice to intranasal and contact infection with mouse hepatitis virus (MHV)-related coronaviruses was tested in infant CD1 mice. One day old mouse pups were inoculated intranasally with respiratory MHV-S, enteric MHV-Y, rat sialodacryoadenitis virus (SDAV), human coronavirus OC43 (HCV-OC43) or bovine coronavirus (BCV). Twenty-four hours later, they were placed in direct contact with age matched sham inoculated pups. Indices of infection in virus inoculated mice included lesions by histopathology and viral antigen by immunoperoxidase histochemistry in brain, lung, liver and intestine at 3 days after inoculation. Indices of infection in contact mice included mortality or seroconversion by 21 days after exposure. Infant mice were susceptible to infection with all five viruses. Transmission by direct contact exposure occurred with MHV and SDAV, but not HCV or BCV. Furthermore, adult mice were not susceptible to infection with HCV. Tissue distribution of lesions and antigen varied markedly among viruses, indicating that they do not induce the same disease as MHV. This study demonstrates that although these coronaviruses are antigenically closely related, they are biologically different viruses and disease patterns in susceptible infant mice can be used to differentiate viruses.     

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O^Tg) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O^Tg mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.     

Reconstructing the origins of human hepatitis viruses

Infections with hepatitis B and C viruses (HBV, HCV) are widespread in human populations throughout the world, and are major causes of chronic liver disease and liver cancer. HBV, HCV and the related hepatitis G virus or GB virus C (referred to here as HGV/GBV-C) are capable of establishing persistent, frequently lifelong infections characterized by high levels of continuous replication. All three viruses show substantial genetic heterogeneity, which has allowed each to be classified into a number of distinct genotypes that have different geographical distributions and associations with different risk groups for infection. Information on their past transmission and epidemiology might be obtained by estimation of the time of divergence of the different genotypes of HCV, HBV and HGV/GBV-C using knowledge of their rates of sequence change. While information on the latter is limited to short observation periods and is therefore subject to considerable error and uncertainty, the relatively recent times of origin for genotype of each virus predicted by this method (HCV, 500-2000 years; HBV, 3000 years; HGV/GBV-C, 200 years) are quite incompatible with their epidemiological distributions in human populations. They also cannot easily be reconciled with the recent evidence for species-associated variants of HBV and HGV/GBV-C in a range of non-human primates. The apparent conservatism of viruses over long periods implied by their epidemiological distributions instead suggests that nucleotide sequence change may be subject to constraints peculiar to viruses with single-stranded genomes, or with overlapping reading frames that defy attempts to reconstruct evolution according to the principles of the 'molecular clock'. Large population sizes and intense selection pressures that optimize fitness may be additional factors that set virus evolution apart from that of their hosts.     

Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C: analysis in transgenic mice and humans

Steatosis is one of the histologic characteristics of chronic hepatitis C and is well reproduced in a transgenic mouse model for hepatocellular carcinoma (HCC) in which the core protein of hepatitis C virus (HCV) plays a pivotal role in inducing steatosis and HCC. In the present study, the lipid composition in the liver of the HCV core gene transgenic mice as well as in those of chronic hepatitis C patients was determined. The concentration of carbon 18 monounsaturated (C18:1) fatty acids, such as oleic and vaccenic acids, which are known to increase membrane fluidity leading to higher cell division rates, significantly increased in the livers of transgenic mice compared to nontransgenic control mice. The concentration of C18:1 fatty acids also significantly increased in the livers of chronic hepatitis C patients compared to subjects without HCV infection. These results suggest that HCV may affect a specific pathway in the lipid metabolism and cause steatosis in the liver.     

Cyclophilin A and viral infections

Cyclophilin A (CyPA) is a peptidyl-prolyl cis/trans isomerase originally identified as the target of the immunosuppressive drug cyclosporine A. A number of reports have demonstrated that CyPA plays a critical role in the successful replication of viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), etc. However, recent studies demonstrated that CyPA also possesses a repressive effect on the replication of some viruses like Influenza A virus and rotavirus. Moreover, CyPA could also regulate host IFN-I response to viral infections. Together, these evidences showed diverse roles of CyPA in viral infection.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.     

Generation of a humanized mouse model with both human immune system and liver cells to model hepatitis C virus infection and liver immunopathogenesis

Establishing a small animal model that accurately recapitulates hepatotropic pathogens, including hepatitis C virus (HCV) infection and immunopathogenesis, is essential for the study of hepatitis virus-induced liver disease and for therapeutics development. This protocol describes our recently developed humanized mouse model for studying HCV and other hepatotropic infections, human immune response and hepatitis and liver fibrosis. The first 5-h stage is the isolation of human liver progenitor and hematopoietic stem cells from fetal liver. Next, AFC8 immunodeficient mice are transplanted with the isolated progenitor/stem cells. This generally takes 2 h. The transplanted mice are then treated for a month with the mouse liver apoptosis-inducing AFC8 dimerizer and left for an additional 2-month period to permit human liver and immune cell growth as well as system reconstitution and development before inoculation with HCV clinical isolates. HCV infection, human immune response and liver disease are observed with high incidence from approximately 2 months after inoculation.