丙型肝炎病毒体外大规模培养技术研究及其在抗丙肝病毒药物筛选中的初步应用
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摘要
丙型肝炎病毒(HCV)是输血后非甲非乙肝炎的主要病因，全世界约有1.7亿人感染HCV，中国有大约4000万的HCV携带者。超过一半的HCV感染者会转化成慢性感染，其中10-20％的慢性感染者会发展成为肝脏疾病如肝硬化，并且最终1-5％的人会发展成为肝细胞癌。目前，对HCV感染的治疗尚无有效的方法，干扰素治疗只对10-20％的病人有效，低于50％的病人对干扰素和病毒唑的联合治疗有反应。由于HCV难以在体外培养，使得HCV疫苗和抗HCV药物的研究进展缓慢。
质粒BRT703'X(NIHJ1)含有HCV全长基因组DNA，将其用限制性内切酶EcoRⅠ和HindⅢ不完全酶切后，回收HCV基因组序列，插入到质粒载体pCOUS-T7的EcoRⅠ和HindⅢ之间，使HCV基因组置于载体的T7启动子和T7终止子控制下，构建质粒pT7HCV。
质粒0.pMKC1A(HCV)含有HCV基因组前体蛋白的开放阅读框(ORF)序列，将其用EcoRⅠ和HindⅢ双酶切以后，插入到质粒PSC59的EcoRⅠ和Stu Ⅰ位点之间，使HCV的ORF置于载体的痘苗病毒晚期启动子控制之下，构建质粒pVHCV。
在本研究中，两个质粒被引入一个体外HCV细胞培养系统中：质粒pT7HCV携带有HCV全长基因组cDNA，并将HCV基因组置于载体的T7启动子和T7终止子的控制下。质粒pVHCV含有HCV前体蛋白开放阅读框(ORF)序列，并且HCV的ORF在载体的痘苗病毒晚期启动子控制下。将两个质粒pT7HCV和pVHCV共转染真核细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒感染此细胞，在一定条件下培养后得到重组
张彦明:丙型肝炎病毒体外大规模培养技术研究及其在抗丙肝病毒药物筛选中的初步应用
一定条件下培养后得到重组HCV。在此基础上，对一系列的培养条件
进行了优化，以期得到最高产量的重组HCV。随后将培养得到的重组HCV
利用蔗糖密度梯度离心进行纯化和鉴定。并且对基因重组干扰素一alb
和病毒哇的抗HCV作用进行了初步的研究，探索本系统应用于筛选抗
HCv药物的可能性。
构建的重组质粒用酶切和核普酸序列测定的方法进行鉴定，结果
证明外源基因序列正确地插入到了载体的相应位置。
将质粒pT7HCv和pvHCv用脂质体LipofeCtaminTM2000共转染BHK21
细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒vTF7一3感染此
细胞，病毒吸附细胞2小时后，换新鲜的10%DMEM培养基在30℃、5
%COZ培养箱中培养2一5天后得到重组HCV。用引物特异性RT一PCR检测
培养上清中HCV的病毒基因组RNA;荧光定量PCR方法测定培养上清中
HCV的拷贝数;Western blotting和间接免疫荧光检测细胞中HCV蛋
白的表达;免疫电镜观察病毒的亚细胞定位。
试验结果显示，在六孔细胞培养板中，培养96小时以后可以在培
养上清中检测到每毫升7xl护个拷贝的重组HCV;RT一PCR结果显示，重
组HCV中含有HCV的基因组RNA;在细胞中检测到了HCV蛋白的表达;
免疫电镜在胞质中观察到直径约SOnm左右的深染色病毒样颗粒。
为了适应HCV疫苗研究的需要，我们对体外培养HCV的条件进行
了优化。以六孔细胞培养板为基础，我们对培养HCV所用的细胞系、转
染细胞所用的质粒量、重组痘苗病毒感染时痘苗病毒和细胞数的比例以
及培养后收获病毒的时间等条件进行了优化。
试验结果显示，选用BHKZ，细胞做为宿主细胞时;细胞用5 pg质粒
pT7HCV和5 pg质粒pVHCV共转染，转染后的细胞以每个细胞5个PFU
的比例用重组痘苗病毒感染:感染后培养96小时的条件下，在细胞培
养上清中可以以相对最少的消耗和最短时间得到最高浓度的HCV。确定
了最终用于HCV生产的优化条件。
将培养得到的重组HCV进行纯化和鉴定。培养结束后，收获细胞
和培养上清，将细胞裂解，低速离心去除细胞碎片，然后用35%的
PEG6O00一NaCI溶液将离心上清进行浓缩，继之以不连续的蔗糖密度梯
度离心。HCV基因组引物特异性RT一PCR鉴定HCV的存在;SDS一PAGE、
第一军医大学博士学位论文
western blotting和免疫荧光抗原片鉴定HCV蛋白的表达;磷钨酸负
染后在电子显微镜下观察纯化后病毒的形态。
结果显示，重组HCV的密度梯度大都集中在约1.159/ml，但在1.09
g/ml处有一小的吸收峰。以重组HCV为模板进行的引物特异性RT一PCR
可以扩增出HCV的5’一UTR、核心蛋白、NS。蛋白和3’UTR基因片段，显
示在重组HCV中包含有全长的HCV基因组;SnS一PAGE、Western blotting
和免疫荧光抗原片检测到了HCV结构蛋白的表达。纯化的病毒用磷钨酸
负染后在电镜下观察可以看到直径为6Onm的带有6nm棘突的HCV病毒
样颗粒。
以干扰素a(IFN一Q)和病毒哇(RBV)为对象，初步研究了本系统
用于筛选抗丙型肝炎病毒药物的可行性。探索了二者对于体外培养的
HCV早期复制过程的影响。
在BHKZ，细胞经过质粒pT7HCV和pVHCv转染并且被重组痘苗病
毒vTF7一3感染后，加入药物基因重组干扰素Q一lb(rIFN一Q lb)和RBV，
培养过程中每隔24小时取样检测上清中HCV的拷贝数。结果显示，rl FN-
Qlb直接抑制HCV在体外的早期复制，并且抑制效应呈剂量依赖性。
RBV在最初的48小时对HCV复制有明显的抑制作用，但是后期的效果
不明显，并且药物浓度对于HCV复制的影响关系很大。说明rIFN一alb
对于HCV的作用除调节机
Ph.D. candidate Zhang Yan-ming Supervisor Li Ming
The hepatitis C virus (HCV) is a major causative agent for most posttransfusion non-A, non-B hepatitis case. There are about 170 million carriers of hepatitis C virus in the world today and about 40 million in China alone. More than half of persons infected with HCV can progress to chronic infection, and of the chronically infected 10-20% will develop complications of chronic liver disease such as liver cirrorhosis and 1-5% will develop hepatocellular carcinoma. Unfortunately, only 10-20% of patients infected by HCV are respond to interferon alone, therapy of IFN- a combined with ribavirin is effective in only about 50% infected persons. The lack of tissue-culture system for HCV replication has limited the development of vaccine for HCV and new treatment.
We propose a cell culture system to produce HCV in vitro. In this system, we constructed two plasmids: pT7HCV contains the HCV genomic RNA-coding region between an upstream T7 promoter and a downstream T7 terminator; pVHCV contains the HCV polyprotein open reading frame (ORF) that is linked to the vaccinia late promoter of vector. The two plasmids were co-transfected into eucaryotic cells, the cells then were infected by recombinant vaccinia virus vTF7-3 which contain a T7 RNA polymerase gene under the control of a vaccinia promoter. After cultured, recombinant HCV can be produced in the cells. We optimized the culture condition of the cells to get the highest production of recombinant HCV. Then we separate HCV by discontinuous sucrose density-gradient ultra-centrifugation. We also investigated effect of interferon and rebavirin
on HCV early replication stage in this HCV culture system.
Plasmid pBRT703'X(NIHJl) containing the full-length HCV genomic cDNA, was incompletely digested with HindIII and EcoR I , then HCV cDNA was reclaimed and inserted into vector pOCUS-T7 (plasmid pOCUS containing a T7 promoter and a T7 terminator) between the EcoR I and Hindlll sites, resulting insertion of the HCV sequence between the bacteriophage T7 promoter and T7 terminator sequences to create plasmid pT7HCV.
Plasmid O. pMKC1A (HCV) containing the HCV polyprotein open reading frame was cut with EcoR I and Hindlll and inserted into PSC59 between the EcoR I and Stu I sites to generate plasmid pVHCV, which contained the HCV polyprotein open reading frame (ORF) linked to a vaccinia late promoter.
Restriction enzyme digestion and DNA sequencing confirm the correct sequences of plasmid pT7HCV and pVHCV.
Cells were then transfected with pT7HCV plus pVHCV using the LipofectaminTM2000 Reagent followed by infection with vTF7-3 vaccinia viruses. Then cells were inoculated two hours for absorption of virus. The inoculum was then removed and the cells were cultured in fresh Dulbecco's modified Eagle's medium (DMEM)with 10% fetal calf serum(FCS) in a 30 "C and 5%CO2 incubator for 2-5 days. Genomic RNA of HCV in the supernatants was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR), the titer of HCV was examined by Fluorescent Quantitative PCR (FQ-PCR), HCV proteins were tested by Indirect Immunofluorescence and Western blot analysis and the virus was observed under Immunoelectron microscopy to locate the virus in cell.
As the result of experiment showed, after 96 hours of incubation in 6-well plates, 7 106 copies of HCV can be produced in the supernatants. HCV genomic RNA was amplified by RT-PCR, and structural and nonstructural proteins of HCV can be detected by immunofluorescence and Western blot. Virus-like particles of about 50nm in diameter were observed
under immuno-electron microscopy.
We optimized the culture condition of HCV for the purpose of produce HCV vaccine. We selected appropriate cell line, determine quantity of plasmid for transfection and choose the best concentration of recombinant vaccinia virus to produce the most abundance HCV in supernatant. The copies of HCV genomic RNA in supernatant was detected by FQ-RT-PCR and used as guideline to determine the condition of HCV culture.
Th
质粒BRT703'X(NIHJ1)含有HCV全长基因组DNA，将其用限制性内切酶EcoRⅠ和HindⅢ不完全酶切后，回收HCV基因组序列，插入到质粒载体pCOUS-T7的EcoRⅠ和HindⅢ之间，使HCV基因组置于载体的T7启动子和T7终止子控制下，构建质粒pT7HCV。
质粒0.pMKC1A(HCV)含有HCV基因组前体蛋白的开放阅读框(ORF)序列，将其用EcoRⅠ和HindⅢ双酶切以后，插入到质粒PSC59的EcoRⅠ和Stu Ⅰ位点之间，使HCV的ORF置于载体的痘苗病毒晚期启动子控制之下，构建质粒pVHCV。
在本研究中，两个质粒被引入一个体外HCV细胞培养系统中：质粒pT7HCV携带有HCV全长基因组cDNA，并将HCV基因组置于载体的T7启动子和T7终止子的控制下。质粒pVHCV含有HCV前体蛋白开放阅读框(ORF)序列，并且HCV的ORF在载体的痘苗病毒晚期启动子控制下。将两个质粒pT7HCV和pVHCV共转染真核细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒感染此细胞，在一定条件下培养后得到重组
张彦明:丙型肝炎病毒体外大规模培养技术研究及其在抗丙肝病毒药物筛选中的初步应用
一定条件下培养后得到重组HCV。在此基础上，对一系列的培养条件
进行了优化，以期得到最高产量的重组HCV。随后将培养得到的重组HCV
利用蔗糖密度梯度离心进行纯化和鉴定。并且对基因重组干扰素一alb
和病毒哇的抗HCV作用进行了初步的研究，探索本系统应用于筛选抗
HCv药物的可能性。
构建的重组质粒用酶切和核普酸序列测定的方法进行鉴定，结果
证明外源基因序列正确地插入到了载体的相应位置。
将质粒pT7HCv和pvHCv用脂质体LipofeCtaminTM2000共转染BHK21
细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒vTF7一3感染此
细胞，病毒吸附细胞2小时后，换新鲜的10%DMEM培养基在30℃、5
%COZ培养箱中培养2一5天后得到重组HCV。用引物特异性RT一PCR检测
培养上清中HCV的病毒基因组RNA;荧光定量PCR方法测定培养上清中
HCV的拷贝数;Western blotting和间接免疫荧光检测细胞中HCV蛋
白的表达;免疫电镜观察病毒的亚细胞定位。
试验结果显示，在六孔细胞培养板中，培养96小时以后可以在培
养上清中检测到每毫升7xl护个拷贝的重组HCV;RT一PCR结果显示，重
组HCV中含有HCV的基因组RNA;在细胞中检测到了HCV蛋白的表达;
免疫电镜在胞质中观察到直径约SOnm左右的深染色病毒样颗粒。
为了适应HCV疫苗研究的需要，我们对体外培养HCV的条件进行
了优化。以六孔细胞培养板为基础，我们对培养HCV所用的细胞系、转
染细胞所用的质粒量、重组痘苗病毒感染时痘苗病毒和细胞数的比例以
及培养后收获病毒的时间等条件进行了优化。
试验结果显示，选用BHKZ，细胞做为宿主细胞时;细胞用5 pg质粒
pT7HCV和5 pg质粒pVHCV共转染，转染后的细胞以每个细胞5个PFU
的比例用重组痘苗病毒感染:感染后培养96小时的条件下，在细胞培
养上清中可以以相对最少的消耗和最短时间得到最高浓度的HCV。确定
了最终用于HCV生产的优化条件。
将培养得到的重组HCV进行纯化和鉴定。培养结束后，收获细胞
和培养上清，将细胞裂解，低速离心去除细胞碎片，然后用35%的
PEG6O00一NaCI溶液将离心上清进行浓缩，继之以不连续的蔗糖密度梯
度离心。HCV基因组引物特异性RT一PCR鉴定HCV的存在;SDS一PAGE、
第一军医大学博士学位论文
western blotting和免疫荧光抗原片鉴定HCV蛋白的表达;磷钨酸负
染后在电子显微镜下观察纯化后病毒的形态。
结果显示，重组HCV的密度梯度大都集中在约1.159/ml，但在1.09
g/ml处有一小的吸收峰。以重组HCV为模板进行的引物特异性RT一PCR
可以扩增出HCV的5’一UTR、核心蛋白、NS。蛋白和3’UTR基因片段，显
示在重组HCV中包含有全长的HCV基因组;SnS一PAGE、Western blotting
和免疫荧光抗原片检测到了HCV结构蛋白的表达。纯化的病毒用磷钨酸
负染后在电镜下观察可以看到直径为6Onm的带有6nm棘突的HCV病毒
样颗粒。
以干扰素a(IFN一Q)和病毒哇(RBV)为对象，初步研究了本系统
用于筛选抗丙型肝炎病毒药物的可行性。探索了二者对于体外培养的
HCV早期复制过程的影响。
在BHKZ，细胞经过质粒pT7HCV和pVHCv转染并且被重组痘苗病
毒vTF7一3感染后，加入药物基因重组干扰素Q一lb(rIFN一Q lb)和RBV，
培养过程中每隔24小时取样检测上清中HCV的拷贝数。结果显示，rl FN-
Qlb直接抑制HCV在体外的早期复制，并且抑制效应呈剂量依赖性。
RBV在最初的48小时对HCV复制有明显的抑制作用，但是后期的效果
不明显，并且药物浓度对于HCV复制的影响关系很大。说明rIFN一alb
对于HCV的作用除调节机
Ph.D. candidate Zhang Yan-ming Supervisor Li Ming
The hepatitis C virus (HCV) is a major causative agent for most posttransfusion non-A, non-B hepatitis case. There are about 170 million carriers of hepatitis C virus in the world today and about 40 million in China alone. More than half of persons infected with HCV can progress to chronic infection, and of the chronically infected 10-20% will develop complications of chronic liver disease such as liver cirrorhosis and 1-5% will develop hepatocellular carcinoma. Unfortunately, only 10-20% of patients infected by HCV are respond to interferon alone, therapy of IFN- a combined with ribavirin is effective in only about 50% infected persons. The lack of tissue-culture system for HCV replication has limited the development of vaccine for HCV and new treatment.
We propose a cell culture system to produce HCV in vitro. In this system, we constructed two plasmids: pT7HCV contains the HCV genomic RNA-coding region between an upstream T7 promoter and a downstream T7 terminator; pVHCV contains the HCV polyprotein open reading frame (ORF) that is linked to the vaccinia late promoter of vector. The two plasmids were co-transfected into eucaryotic cells, the cells then were infected by recombinant vaccinia virus vTF7-3 which contain a T7 RNA polymerase gene under the control of a vaccinia promoter. After cultured, recombinant HCV can be produced in the cells. We optimized the culture condition of the cells to get the highest production of recombinant HCV. Then we separate HCV by discontinuous sucrose density-gradient ultra-centrifugation. We also investigated effect of interferon and rebavirin
on HCV early replication stage in this HCV culture system.
Plasmid pBRT703'X(NIHJl) containing the full-length HCV genomic cDNA, was incompletely digested with HindIII and EcoR I , then HCV cDNA was reclaimed and inserted into vector pOCUS-T7 (plasmid pOCUS containing a T7 promoter and a T7 terminator) between the EcoR I and Hindlll sites, resulting insertion of the HCV sequence between the bacteriophage T7 promoter and T7 terminator sequences to create plasmid pT7HCV.
Plasmid O. pMKC1A (HCV) containing the HCV polyprotein open reading frame was cut with EcoR I and Hindlll and inserted into PSC59 between the EcoR I and Stu I sites to generate plasmid pVHCV, which contained the HCV polyprotein open reading frame (ORF) linked to a vaccinia late promoter.
Restriction enzyme digestion and DNA sequencing confirm the correct sequences of plasmid pT7HCV and pVHCV.
Cells were then transfected with pT7HCV plus pVHCV using the LipofectaminTM2000 Reagent followed by infection with vTF7-3 vaccinia viruses. Then cells were inoculated two hours for absorption of virus. The inoculum was then removed and the cells were cultured in fresh Dulbecco's modified Eagle's medium (DMEM)with 10% fetal calf serum(FCS) in a 30 "C and 5%CO2 incubator for 2-5 days. Genomic RNA of HCV in the supernatants was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR), the titer of HCV was examined by Fluorescent Quantitative PCR (FQ-PCR), HCV proteins were tested by Indirect Immunofluorescence and Western blot analysis and the virus was observed under Immunoelectron microscopy to locate the virus in cell.
As the result of experiment showed, after 96 hours of incubation in 6-well plates, 7 106 copies of HCV can be produced in the supernatants. HCV genomic RNA was amplified by RT-PCR, and structural and nonstructural proteins of HCV can be detected by immunofluorescence and Western blot. Virus-like particles of about 50nm in diameter were observed
under immuno-electron microscopy.
We optimized the culture condition of HCV for the purpose of produce HCV vaccine. We selected appropriate cell line, determine quantity of plasmid for transfection and choose the best concentration of recombinant vaccinia virus to produce the most abundance HCV in supernatant. The copies of HCV genomic RNA in supernatant was detected by FQ-RT-PCR and used as guideline to determine the condition of HCV culture.
Th
引文
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14. J. Sambrook, E. F. Fritsch, T. Maniatis. Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, 1989.
15. Brown E.A. et.al secondary structure of the 5 nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acid Res1992,20:5041-5045
16. Fukushia S et.al. The sequence element of the internal ribosome entry site and a 25 kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA. J Virol. 1997,71:1662-1666
17．曾毅 艾滋病毒及其有关病毒 南开大学出版社1999 210-211
18．刘玉斌，苟仕金主编，动物免疫学试验技术吉林科学技术出版社1989年
19. Ali, N., K. D. Tardif, and A. Siddiqui. 2002. Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon. J. Virol. 76: 12001-12007.
20. 20. Alter, M. J., D. Kruszon-Moran, O. Nainan, G. McQuillan, F. Gao, L. A. Moyer, R. A. Kaslow, and H. S. Margolis. 1999. The prevalence of hepatitis C virus infection in the United States 1988-1994. N. Engl. J. Med. 341: 556-562.
21. Altschuler, M., R. Tritz, and A. Hampel. A method for generating transcripts with defined 5' and 3' termini by autolytic processing. Gene.122: 85-90.
22. Baumert, T. F.,S. Ito, D. T. Wong, and T. J. Liang. 1998. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J. Virol.72:3827-3836.
23. Blanchard, E., D. Brand, S. Trassard, A. Goudeau, and P. Roingeard. 2002.
Hepatitis C virus-like particle morphogenesis. J Virol. 76: 4073-4079.
24. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-1974.
25. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2002. Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication. J. Virol. 76: 13001-13014.
26. Blight, K. J., J. A. McKeating, J. Marcotrigiano, and C. M. Rice. 2003. Efficient Replication of Hepatitis C Virus Genotype la RNAs in Cell Culture. J. Virol. 77: 3181-3190.
27. Chung, R. T., W. He, A. Saquib, A. M. Contreras, R. J. Xavier, A. Chawla, T. C. Wang, and E. V. Schmidt. 2001. Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system.Proc Natl Acad Sci U S A. 98: 9847-9852.
28. Krieger, N., V. Lohmann, and R. Bartenschlager. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J. Virol. 75:4614-4624.
29. Mizuno, M., G. Yamada, T. Tanaka, K. Shimotohno, M. Takatani, and T. Tsuji. 1995. Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome. Gastroenterology. 109: 1933-1940.
30. Pietschmann, T., Lohmann, V. Kaul, A. Krieger, N. Rinck, G. Rutter, G. Strand, D. and R. Bartenschlager. 2002. Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture. J. Virol. 76: 4008-4021.
31. Selby, M. J., Q. L. Choo, K. Berger, G. Kuo, E. Glazer, M. Eckart, C. Lee, D. Chien, C. Kuo, and M. Houghton. 1993. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J Gen Virol. 74:1103-1113.
32. Wellnitz, S., B. Klumpp, H. Barth, S. Ito, E. Depla, J. Dubuisson, H. E. Blum, and T. F. Baumert. 2002. Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines. J Virol.
76:1181-93.
33. YI, M., and S. M. LEMON. 2003. Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA. 9:331-345.
34. Yoo, B. J., M. J. Selby, J. Choe, B. S. Suh, S. H. Choi, J. S. Joh, G. J. Nuovo, H. S. Lee, M. Houghton, and J. H. Han. 1995. Tmnsfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol. 69: 32-38.
35. 11. Sells MA, Zelent AZ, Shvartsman M,et.al. Replicate intermediate of hepatitis B virus in HepG2 cells that produce infection virions. J Virol 1988; 62:2836-2844
36. 12. Sureau C, Romet J L, Mullins J, et.al Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 1986; 47:37-47
37．宋志强，郝飞，马巧玉，王宇明．丙型肝炎病毒体外感染人肝癌细胞株
38．HepG-2的研究，中华实验验和临床病毒学杂志，2003；17(1)：77-80
39. Choo Q.L,G. Kuo, A.J Weiner et.al Isolation of a eDNA clone derived from a blood-borne nonA-nonB viral hepatitis genome. Science 1989, 244:359-362
40．F．奥斯伯，R．E．金斯敦，J．G．塞德曼等著，颜子颍，王海林译，精遍分子生物学实验指南，2001，科学出版社，286-287
41．傅继华 病毒学实用实验技术 山东科学技术出版社2000
42. Naoki F, Massahiko K and Satoshi I et.al Paraformaldehyde protects of hepatitis C virus particles during ultracentrifugation. J Med Viro, 2001,63:108-116
43. Homas F B, Susumu I and David T et.al. Hepatitis C virus structural proteins assemble into virus-like particles in insect cells. J Viro, 1998, 72:3827-3836
44．林学颜 免疫学基础 福建科技出版社1980：155-158
45. Sambrook J, Fritsch EF,Maniafis T, et.al Molecular cloning, a laboratory manual [M]. 2nd ed, New York: Cold Spring Harbor Laboratory, 1989
46. Reginald F C, Ania O and Jim A. Analysis of antigenicity and topology of glycoprotein present on recombinant hepatitis C virus-like particles. J Viro, 2002, 76:7672-7682
47. Bradely DW, Krawczynski K, Cook EH Jr et al. Enterically transmitted non-A,non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease- associated 27-to34-nm virus-like particles. Proc Natl Acad Sci USA, 1987; 84(17): 6277-6281
48. Miyamoto H, Okamoto H and Sato K et.al Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient eentrifugation and the polymerase chain reaction, J Gen Virol 1992, 73:715-718
49. Hijikata M, Shimizu Yk and Kato H et.al. Equilibrium centrifugation studies if hepatitis C virus: evidence for circulating immune complexes. J Viro, 1993, 67:1953-1958
50. Kaito M, Watanabes S and Tsukiyauma-kohara K et.al. Hepatitis C virus particle defected by immumoelectron microscopic study. J Gen Viro 1994, 75:1755-1760
51. Kanto T, Hayashi N and Hagiwara H et.al. Buoyant density of hepatitis C virus recovered from infected hosts: two different features in sucrose equilibrium density-gradient centrifugation related to degree of liver inflammation. Hepatology, 1994, 19:296-302
52. Choo SH, So HS, Cho JM et al. Association of hepatitis C virus particles with immuno- globulin: a mechanism for persistent infection. J Gen Virol, 1995; 76(9): 2337-2341
53. Burk KH, Cabral GA, Dreesman GR et al. Ultrastructural change and virus-like particles localized in liver hepatocytes of chimpanzees infected with non-A, non-B hepatitis. J Med Virol, 1981;7(1):1-19
54. Hantz O, Vitvitski L, Trepo C. Non-A, non-B hepatitis :identification of hepatitis-B-like virus particles in serum and liver. J Med Virol, 1980; 5(1): 73-88
55. Bosman C, Valli MB, Bertolini Let al. Detection of vires-like particles in liver biopsies from HCV-infected pationts. Res Virol, 1998; 69(3): 731-741
56. Shimizu YK, Feinstone SM, Kohara Met al. Hepatitis C virus: detection of intmcellular virus particles by electron microscopy. Hepatology, 1996; 23(2): 205-206
57. Baumert TF, Ito S, Wong DT et al. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol, 1998; 72(5):3827-3836
58. Isaacs, A. and J, Lindenmann. 1957. Virus interference.1.The interferon. Proc. R. Soc. London Scr, B 147:258-267
59．金奇等 医学分子病毒学2001科学出版社
60. Samuel C E. Antivirial actions of interferons. Clinical microbiology Reviews. 14:118-809
61．黄建生 任大明 丙型肝炎2000年人民军医出版社
62. Reichen J, Bianchi L, Buhler H et.al. Fixed versus titraed inrerferon-α 2b in chronic hepatitis C. A randomized controlled multicenter trial. The Swiss Association for the study of the Liver. J Hepatol, 1996;25(3):275-280
63. Bortolotti F, Giacchino R, Vajro P et.al. Recombinant interferon-alpha therapy in children with chronic hepatitis C. Hepatology, 1995; 22(6): 1623-1630
64. Jouet P, Roudot-Thoraval F, Dhumeaux D et.al. Comparative efficacy of interferon-alpha in cirrhotic and noncirrhotic patients with non-A,non-B C hepatitis. Le Group Francais pour 1' Etude du Traitement des Hepatitis Chroniques NANB/C. Gastroenterology, 1994; 106(3): 286-671
65．陈士俊，林颖敏，马继顺等，重组干扰素-α 2b不同疗法治疗慢性丙型肝炎，中华传染病杂志，1997；15(3)：167-168
66．史冬梅，高健，陆志檬等，两种剂量重组干扰素-α 2a治疗慢性丙型肝炎的疗效观察，中华传染病杂志，1997；15(2)：110-111
67．徐道振，牛润英，林佳佳，国产重组干抗素-α1b治疗慢牲丙型肝炎
追访2年的疗效观察，中华消化杂志，1994；14(1)：5-7
68. Orito E, Mizokami M, Nakano T te.al. Serum hepatitis C virus RNA level as a predictor of subsequent response to interferon-alpha therapy in Japanese patients with chronic hepatitis C. J Med Virol, 1994; 44(4): 40
69．张复春，洗超，菜晓莉等，干扰素治疗慢性丙型肝炎疗效与血清HCVRNA水平关系的研究，中华肝脏病杂志，1997；5(4)：211-213
70. Koskinase J, Tibbs C, Saleh M G et.al. Effects of ribavirin on intrahepatic and extrahepatic expression of hepatitis C virus in interferon non-responsive patients. J Med Virol, 1995; 45(1): 29-31
71. Zoulim F, Haem J, Ahmed SS et.al. Ribavirin mono-therapy in patients with chronic hepatitis C: a retrospective study of 95 patients. J Viral Hepat, 1998;5(3): 193-199
72. Di Bisceglie AM, Conjeevaram HS, Fried MW et.al Ribavirin as therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. Ann Intern Med, 1995; 123(12): 897-903
73. Hoofnagle JH, Lau D, Conjeeram H et.al. Prolonged therapy of chronic hepatitis C with ribavirin. J Viral Hepat, 1996;3(5): 247
74．中国医学科学院流行病防治研究所编 常见病毒病试验技术1978科学出版社
2．国际丙型肝炎共识研讨会文献，巴黎，1999年2月26日至28
3. Tong, M.J, N.S. EI-Farra, A.R. Reikes, and R.L.Co, Clinical outcomes after transfusion- associated hepatitis C, N Engl. J. Med. 1995, 332:1463-1466
4．丙型肝炎防治指南，中华医学会肝病学分会和传染病学与寄生虫病学分会，2004
5. Lanford RE, Sureau C, Jacob JR. Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR。Virology. 1994; 202(2):606-14
6. Ikeda M, Sugiyama K, Mizutani T. Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus. Virus Res. 1998; 56(2): 157-67
7. Ozeki T, Hikiji K, Funakoshi K. Infection of Chang cells with hepatitis C virus using hepatic biopsy specimens from patients with chronic hepatitis (type C). Int J Exp Pathol. 1992;73(1):1-8.
8. Carloni G, Iacovacci S, Sargiacomo M. Susceptibility of human liver cell cultures to hepatitis C virus infection. Arch Virol Suppl. 1993;8:31-39
9. Shimizu YK, Purcell RH, Yoshikura H. Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro. Proc Natl Acad Sci U S A. 1993; 90(13): 6037-6041.
10. Nakajima N, Hijikata M, Yoshikura H, Shimizu YK. Characterization of long-term cultures of hepatitis C virus, J Virol. 1996; 70(5):3325-3329.
11. Sung VM, Shimodaira S, Doughty AL, et.al. Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol. 2003; 77(3):2134-2146
12. Yoo BJ, Selby MJ, Choe J et.al. Transfection of a differentiated human
hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus(HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol, 1995; 69(1):32-39
13. Selby MJ, Choo QL, Berger K, et.al. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J Gen. Virol. 1993; 74 (6): 1103-1113.
14. J. Sambrook, E. F. Fritsch, T. Maniatis. Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, 1989.
15. Brown E.A. et.al secondary structure of the 5 nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acid Res1992,20:5041-5045
16. Fukushia S et.al. The sequence element of the internal ribosome entry site and a 25 kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA. J Virol. 1997,71:1662-1666
17．曾毅 艾滋病毒及其有关病毒 南开大学出版社1999 210-211
18．刘玉斌，苟仕金主编，动物免疫学试验技术吉林科学技术出版社1989年
19. Ali, N., K. D. Tardif, and A. Siddiqui. 2002. Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon. J. Virol. 76: 12001-12007.
20. 20. Alter, M. J., D. Kruszon-Moran, O. Nainan, G. McQuillan, F. Gao, L. A. Moyer, R. A. Kaslow, and H. S. Margolis. 1999. The prevalence of hepatitis C virus infection in the United States 1988-1994. N. Engl. J. Med. 341: 556-562.
21. Altschuler, M., R. Tritz, and A. Hampel. A method for generating transcripts with defined 5' and 3' termini by autolytic processing. Gene.122: 85-90.
22. Baumert, T. F.,S. Ito, D. T. Wong, and T. J. Liang. 1998. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J. Virol.72:3827-3836.
23. Blanchard, E., D. Brand, S. Trassard, A. Goudeau, and P. Roingeard. 2002.
Hepatitis C virus-like particle morphogenesis. J Virol. 76: 4073-4079.
24. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-1974.
25. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2002. Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication. J. Virol. 76: 13001-13014.
26. Blight, K. J., J. A. McKeating, J. Marcotrigiano, and C. M. Rice. 2003. Efficient Replication of Hepatitis C Virus Genotype la RNAs in Cell Culture. J. Virol. 77: 3181-3190.
27. Chung, R. T., W. He, A. Saquib, A. M. Contreras, R. J. Xavier, A. Chawla, T. C. Wang, and E. V. Schmidt. 2001. Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system.Proc Natl Acad Sci U S A. 98: 9847-9852.
28. Krieger, N., V. Lohmann, and R. Bartenschlager. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J. Virol. 75:4614-4624.
29. Mizuno, M., G. Yamada, T. Tanaka, K. Shimotohno, M. Takatani, and T. Tsuji. 1995. Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome. Gastroenterology. 109: 1933-1940.
30. Pietschmann, T., Lohmann, V. Kaul, A. Krieger, N. Rinck, G. Rutter, G. Strand, D. and R. Bartenschlager. 2002. Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture. J. Virol. 76: 4008-4021.
31. Selby, M. J., Q. L. Choo, K. Berger, G. Kuo, E. Glazer, M. Eckart, C. Lee, D. Chien, C. Kuo, and M. Houghton. 1993. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J Gen Virol. 74:1103-1113.
32. Wellnitz, S., B. Klumpp, H. Barth, S. Ito, E. Depla, J. Dubuisson, H. E. Blum, and T. F. Baumert. 2002. Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines. J Virol.
76:1181-93.
33. YI, M., and S. M. LEMON. 2003. Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA. 9:331-345.
34. Yoo, B. J., M. J. Selby, J. Choe, B. S. Suh, S. H. Choi, J. S. Joh, G. J. Nuovo, H. S. Lee, M. Houghton, and J. H. Han. 1995. Tmnsfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol. 69: 32-38.
35. 11. Sells MA, Zelent AZ, Shvartsman M,et.al. Replicate intermediate of hepatitis B virus in HepG2 cells that produce infection virions. J Virol 1988; 62:2836-2844
36. 12. Sureau C, Romet J L, Mullins J, et.al Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 1986; 47:37-47
37．宋志强，郝飞，马巧玉，王宇明．丙型肝炎病毒体外感染人肝癌细胞株
38．HepG-2的研究，中华实验验和临床病毒学杂志，2003；17(1)：77-80
39. Choo Q.L,G. Kuo, A.J Weiner et.al Isolation of a eDNA clone derived from a blood-borne nonA-nonB viral hepatitis genome. Science 1989, 244:359-362
40．F．奥斯伯，R．E．金斯敦，J．G．塞德曼等著，颜子颍，王海林译，精遍分子生物学实验指南，2001，科学出版社，286-287
41．傅继华 病毒学实用实验技术 山东科学技术出版社2000
42. Naoki F, Massahiko K and Satoshi I et.al Paraformaldehyde protects of hepatitis C virus particles during ultracentrifugation. J Med Viro, 2001,63:108-116
43. Homas F B, Susumu I and David T et.al. Hepatitis C virus structural proteins assemble into virus-like particles in insect cells. J Viro, 1998, 72:3827-3836
44．林学颜 免疫学基础 福建科技出版社1980：155-158
45. Sambrook J, Fritsch EF,Maniafis T, et.al Molecular cloning, a laboratory manual [M]. 2nd ed, New York: Cold Spring Harbor Laboratory, 1989
46. Reginald F C, Ania O and Jim A. Analysis of antigenicity and topology of glycoprotein present on recombinant hepatitis C virus-like particles. J Viro, 2002, 76:7672-7682
47. Bradely DW, Krawczynski K, Cook EH Jr et al. Enterically transmitted non-A,non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease- associated 27-to34-nm virus-like particles. Proc Natl Acad Sci USA, 1987; 84(17): 6277-6281
48. Miyamoto H, Okamoto H and Sato K et.al Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient eentrifugation and the polymerase chain reaction, J Gen Virol 1992, 73:715-718
49. Hijikata M, Shimizu Yk and Kato H et.al. Equilibrium centrifugation studies if hepatitis C virus: evidence for circulating immune complexes. J Viro, 1993, 67:1953-1958
50. Kaito M, Watanabes S and Tsukiyauma-kohara K et.al. Hepatitis C virus particle defected by immumoelectron microscopic study. J Gen Viro 1994, 75:1755-1760
51. Kanto T, Hayashi N and Hagiwara H et.al. Buoyant density of hepatitis C virus recovered from infected hosts: two different features in sucrose equilibrium density-gradient centrifugation related to degree of liver inflammation. Hepatology, 1994, 19:296-302
52. Choo SH, So HS, Cho JM et al. Association of hepatitis C virus particles with immuno- globulin: a mechanism for persistent infection. J Gen Virol, 1995; 76(9): 2337-2341
53. Burk KH, Cabral GA, Dreesman GR et al. Ultrastructural change and virus-like particles localized in liver hepatocytes of chimpanzees infected with non-A, non-B hepatitis. J Med Virol, 1981;7(1):1-19
54. Hantz O, Vitvitski L, Trepo C. Non-A, non-B hepatitis :identification of hepatitis-B-like virus particles in serum and liver. J Med Virol, 1980; 5(1): 73-88
55. Bosman C, Valli MB, Bertolini Let al. Detection of vires-like particles in liver biopsies from HCV-infected pationts. Res Virol, 1998; 69(3): 731-741
56. Shimizu YK, Feinstone SM, Kohara Met al. Hepatitis C virus: detection of intmcellular virus particles by electron microscopy. Hepatology, 1996; 23(2): 205-206
57. Baumert TF, Ito S, Wong DT et al. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol, 1998; 72(5):3827-3836
58. Isaacs, A. and J, Lindenmann. 1957. Virus interference.1.The interferon. Proc. R. Soc. London Scr, B 147:258-267
59．金奇等 医学分子病毒学2001科学出版社
60. Samuel C E. Antivirial actions of interferons. Clinical microbiology Reviews. 14:118-809
61．黄建生 任大明 丙型肝炎2000年人民军医出版社
62. Reichen J, Bianchi L, Buhler H et.al. Fixed versus titraed inrerferon-α 2b in chronic hepatitis C. A randomized controlled multicenter trial. The Swiss Association for the study of the Liver. J Hepatol, 1996;25(3):275-280
63. Bortolotti F, Giacchino R, Vajro P et.al. Recombinant interferon-alpha therapy in children with chronic hepatitis C. Hepatology, 1995; 22(6): 1623-1630
64. Jouet P, Roudot-Thoraval F, Dhumeaux D et.al. Comparative efficacy of interferon-alpha in cirrhotic and noncirrhotic patients with non-A,non-B C hepatitis. Le Group Francais pour 1' Etude du Traitement des Hepatitis Chroniques NANB/C. Gastroenterology, 1994; 106(3): 286-671
65．陈士俊，林颖敏，马继顺等，重组干扰素-α 2b不同疗法治疗慢性丙型肝炎，中华传染病杂志，1997；15(3)：167-168
66．史冬梅，高健，陆志檬等，两种剂量重组干扰素-α 2a治疗慢性丙型肝炎的疗效观察，中华传染病杂志，1997；15(2)：110-111
67．徐道振，牛润英，林佳佳，国产重组干抗素-α1b治疗慢牲丙型肝炎
追访2年的疗效观察，中华消化杂志，1994；14(1)：5-7
68. Orito E, Mizokami M, Nakano T te.al. Serum hepatitis C virus RNA level as a predictor of subsequent response to interferon-alpha therapy in Japanese patients with chronic hepatitis C. J Med Virol, 1994; 44(4): 40
69．张复春，洗超，菜晓莉等，干扰素治疗慢性丙型肝炎疗效与血清HCVRNA水平关系的研究，中华肝脏病杂志，1997；5(4)：211-213
70. Koskinase J, Tibbs C, Saleh M G et.al. Effects of ribavirin on intrahepatic and extrahepatic expression of hepatitis C virus in interferon non-responsive patients. J Med Virol, 1995; 45(1): 29-31
71. Zoulim F, Haem J, Ahmed SS et.al. Ribavirin mono-therapy in patients with chronic hepatitis C: a retrospective study of 95 patients. J Viral Hepat, 1998;5(3): 193-199
72. Di Bisceglie AM, Conjeevaram HS, Fried MW et.al Ribavirin as therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. Ann Intern Med, 1995; 123(12): 897-903
73. Hoofnagle JH, Lau D, Conjeeram H et.al. Prolonged therapy of chronic hepatitis C with ribavirin. J Viral Hepat, 1996;3(5): 247
74．中国医学科学院流行病防治研究所编 常见病毒病试验技术1978科学出版社