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Volume 22 Issue 1
February 2007

    Yin-ping LU, Bao-ju WANG, Ji-hua DONG, Zhao LIU, Shi-he GUAN, Meng-ji LU and Dong-liang YANG. Construction and Characterization of a Hepatitis B Virus Replicon[J]. Virologica Sinica, 2007, 22(1): 8-13.
    Citation: Yin-ping LU, Bao-ju WANG, Ji-hua DONG, Zhao LIU, Shi-he GUAN, Meng-ji LU, Dong-liang YANG. Construction and Characterization of a Hepatitis B Virus Replicon .VIROLOGICA SINICA, 2007, 22(1) : 8-13.
    shu

    乙型肝炎病毒感染性复制子的构建及其特性

    • 1.

      .430022 武汉，华中科技大学同济医学院附属协和医院病毒研究室卢银平、董继华、刘朝;2.华中科技大学同济医学院附属同济医院临床免疫研究室卢银平、王宝菊、杨东亮;3.德国埃森大学病毒学研究所管世鹤、陆蒙吉

    • 通讯作者： 卢银平, yinpinglu@163.com; 
    • 收稿日期： 2006-07-04
      录用日期： 2006-10-24
    • 摘要： 为了构建乙型肝炎病毒（HBV）感染性复制子表达载体，建立HBV体外复制细胞模型，我们以含HBV双倍基因组的质粒pHBV-dimer为模板，用PCR结合限制性酶切的方法分步将1.3拷贝HBV基因组克隆到pCR2.1载体。将构建的HBV复制子pHBV1.3质粒体外转染Huh7细胞，分析转染后不同时间HBsAg、HBeAg表达及HBV复制中间体和转录子水平，并利用该细胞模型进行阿德福韦抗病毒研究。 结果表明成功构建了HBV感染性复制子表达质粒，该质粒上HBV基因能在Huh7细胞中进行高水平复制、转录和表达，并能在体外短期传代达12天。阿德福韦能体外抑制该HBV复制子的复制，抑制程度依赖于药物浓度。新构建的HBV感染性复制子表达载体可介导高水平病毒复制，有望用于HBV复制机制和抗病毒等方面的研究

    Construction and Characterization of a Hepatitis B Virus Replicon

    • 1.

      Departmant of Virology,Union Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,P. R. China

    • 2.

      Division of Clinical Immunology,Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,P. R. China

    • 3.

      Institute of Virology,Duisburg-Essen University,Essen 45147,Germany

    • Corresponding author: Yin-ping LU, yinpinglu@163.com
    • Received Date: 04 July 2006
      Accepted Date: 24 October 2006

      Fund Project: National Natural Science Foundation No.30271170National Natural Science Foundation No.30170889

    • To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg，HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.
    • 加载中

      1. Dandri M,Volz T K,Lutgehetmann M,et al. 2005. Animal models for the study of HBV replication and its variants [J]. J Clin Virol,34 (suppl 1):S54-62.

      2. Guidotti L G,Matzke B,Schaller H,et al. 1995. High-level hepatitis B virus replication in transgenic mice[J]. J Virol,69:6158-6169.

      3. Guo Y,Guo H,Zhang L,et al. 2005. Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells[J]. J Virol,79: 14392-14403.
          doi: 10.1128/JVI.79.22.14392-14403.2005

      4. Hadziyannis S J,Tassopoμlos N C,Heathcote E J,et al. 2003. Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B[J]. N Engl J Med,348:800-807.
          doi: 10.1056/NEJMoa021812

      5. Maddrey W C. 2000. Hepatitis B: an important public health issue[J]. J Med Virol,61:362-366.
          doi: 10.1002/(ISSN)1096-9071

      6. Marcellin P,Chang T T,Lim S G,et al. 2003. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B[J]. N Engl J Med,348:808-816.
          doi: 10.1056/NEJMoa020681

      7. Park S G,Ryu H M,Lim S O,et al. 2005. Interferon-gamma inhibits hepatitis B virus-induced NF-kappa B activa-tion through nuclear localization of NF-kappa B-inducing kinase[J]. Gastroenterology,128:2042-2053.
          doi: 10.1053/j.gastro.2005.03.002

      8. Sauerbrei A,Schacke M,Schultz U,et al. 2005. Alternative methods for validation of cell culture infection with duck hepatitis B virus [J]. J Virol Methods,129:178-185.
          doi: 10.1016/j.jviromet.2005.05.025

      9. Sells M A,Chen M L,Acs G. 1987. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus[J]. Proc Natl Acad Sci USA,84 :1005-1009.
          doi: 10.1073/pnas.84.4.1005

      10. Villeneuve J P. 2005. The natural history of chronic hepatitis B virus infection[J]. J Clin Virol,34 (suppl 1):S139-142.

      11. Wang C Y,Giambrone J J,Smith B F. 2002. Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR[J]. J Virol Methods,106 :175-184.
          doi: 10.1016/S0166-0934(02)00161-1

      12. Yaginuma K,Shirakata Y,Kobayashi M,et al. 1987. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA[J]. Proc Natl Acad Sci USA,84:2678-2682.
          doi: 10.1073/pnas.84.9.2678

      13. Yang P L,Althage A,Chung J,et al. 2002. Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection[J]. Proc Natl Acad Sci USA,99:13825-13830.
          doi: 10.1073/pnas.202398599

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      Construction and Characterization of a Hepatitis B Virus Replicon

        Corresponding author: Yin-ping LU, yinpinglu@163.com
      • 1. Departmant of Virology,Union Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,P. R. China
      • 2. Division of Clinical Immunology,Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,P. R. China
      • 3. Institute of Virology,Duisburg-Essen University,Essen 45147,Germany
      • Received Date: 04 July 2006
      • Accepted Date: 24 October 2006
      Fund Project:  National Natural Science Foundation No.30271170National Natural Science Foundation No.30170889

      Abstract: To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg，HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.

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