Production of infectious hepatitis C virus of various genotypes in cell cultures
Production of infectious hepatitis C virus of various genotypes in cell cultures
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DOI：
10.1128/jvi.02334-06
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发表时间：
2007-05-01
影响因子：
5.4
通讯作者：
Liang, T. Jake
中科院分区：
文献类型：
--
作者：
Kato, Takanobu;Matsumura, Takuya;Liang, T. Jake
一种独特的丙型肝炎病毒株JFH-1已被证明能在细胞培养中有效复制，产生传染性丙型肝炎病毒。我们以前开发了一种DNA表达系统，该系统包含了两个自切割核酶侧翼的丙型肝炎病毒cDNA，以在细胞培养中产生丙型肝炎病毒颗粒。在本研究中，我们在丙型肝炎病毒核酶系统中产生了不同基因型的丙型肝炎病毒颗粒，包括1a(1177)、1b(CG1b)和2a(J6和JFH-1)。构建的载体还含有分泌的碱性磷酸酶基因，以控制转染效率和培养条件的影响。转染Huh7细胞系Huh7.5.1后，通过检测培养上清液中的丙型肝炎病毒RNA和核心抗原，证实了丙型肝炎病毒的持续复制和分泌。H77、CG1b和J6株的丙型肝炎病毒复制水平相当，而JFH-1株的复制水平明显高于其他株。将JFH-1转基因细胞的培养液接种于未感染的Huh7.5.1细胞，以评价其体外感染性。免疫后3d用免疫荧光法检测丙型肝炎病毒蛋白。为了评价病毒在体内的感染性，将丙型肝炎病毒基因1b转基因细胞的培养液接种到黑猩猩体内，造成典型的丙型肝炎病毒感染过程。体外和体内扩增的丙型肝炎病毒1b的序列与用于细胞培养的丙型肝炎病毒基因组c DNA序列相同。丙型肝炎病毒不同基因型培养体系的建立，不仅为研究丙型肝炎病毒的复制和致病机制，也为筛选抗病毒药物提供了有价值的工具。
A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including la (1177), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.