Host Cell Permissiveness and Cell Culture Adaptations
Numerous examples in the literature document that infection of primary human liver cells and several cell lines with serum-derived HCV is possible and that the viral genome can be kept in some of these cell lines for up to 2 years (reviewed in ref. 3). Nevertheless, in all cases, HCV replication did not exceed copy numbers of 0.01–0.1 RNA genomes per cell, limiting the usefulness of these systems. Moreover, infectivity of serum-derived virus is variable, and the sequences of the genomes used for inoculation usually are unknown. However, only when virus production from cloned genomes is possible, the full power of reverse genetics, in which distinct mutations introduced into the viral genome can be analyzed for their impact on replication and virus production, can be used. For these reasons, much effort was invested to generate HCV by transfection of cultured cells with cloned viral genomes; however, until very recently (1), convincing success had not been reported. A first major obstacle toward an efficient cell culture system was overcome with the invention of the HCV replicon system (4). It is based on the efficient and autonomous replication of viral “minigenomes” into which a selectable marker was inserted. Soon thereafter, it was found that the efficiency of replicon RNA amplification was determined by cell culture adaptive mutations within the viral proteins and by selection for particular cells that are highly permissive (3). The latter conclusion is based on the observation that removal of the replicon from a cell clone by treatment with IFN or a selective drug frequently results in cell clones that support higher levels of HCV RNA replication as compared to na?ve Huh-7 cells. The underlying reason for the higher permissiveness is largely unknown, but for one particular cell clone, designated Huh7.5 (5), a single point mutation in the dsRNA sensor retinoic acid-inducible gene-I (RIG-I) was found to be involved in higher permissiveness for HCV RNA replication (6). Activation of RIG-I by dsRNA, such as HCV RNA, results in the phosphorylation and nuclear translocation of IFN regulatory factor-3 (IRF-3), activating innate antiviral defenses. This defect, together with the overall very low expression of the exogenous dsRNA sensor Toll-like receptor 3 in Huh-7 cells (7), could explain why HCV replicates so efficiently in Huh7.5 cells. This is the reason why Zhong et al. (2) generated a Huh7.5-derived cell line and used it for their study.

5. Blight, K. J., McKeating, J. A. & Rice, C. M. (2002) J. Virol. 76, 13001–13014. [PMC free article] [PubMed]
6. Sumpter, R., Jr., Loo, Y. M., Foy, E., Li, K., Yoneyama, M., Fujita, T., Lemon, S. M. & Gale, M., Jr. (2005) J. Virol. 79, 2689–2699. [PMC free article] [PubMed]

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The ability to grow HCV in vitro is important for understanding both virologic and immunologic aspects of HCV infections. It has been shown that the JFH1 and the chimeric J6/JFH1 isolate of the 2a genotype of HCV replicate efficiently in Huh7 cells [4,5] and in the highly permissive Huh7.5 and Huh7.5.1 cells derived from the human hepatoma cell line Huh7 [6-9]. Later, production of infectious genotype 1a and 1b viruses [10] was demonstrated in Huh7.5 cells. Further studies showed that the increased permissiveness of Huh7.5 cells results from a mutation (Thr-55-Iso) in the RIG-I gene (retinoic acid-inducible gene I, a DExD/H domain containing RNA helicase, reviewed in [11]) which impairs interferon signaling [12].

6   Blight KJ, McKeating JA, Rice CM: Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.

Journal of Virology 2002, 76:13001-13014. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

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7   Zhong J, Gastaminza P, Cheng GF, Kapadia S, Kato T, Burton DR, et al.: Robust hepatitis C virus infection in vitro.

Proceedings of the National Academy of Sciences of the United States of America 2005, 102:9294-9299. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

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8   Yi M, Villanueva RA, Thomas DL, Wakita T, Lemon SM: Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells.

Proceedings of the National Academy of Sciences of the United States of America 2006, 103:2310-2315. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

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9   Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, et al.: Complete replication of hepatitis C virus in cell culture.

Science 2005, 309:623-626. PubMed Abstract | Publisher Full Text
Huh7.5
Blight, K.J., J.A. McKeating and C.M. Rice, Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol, 2002. 76(24): p. 13001-14.


　Huh7细胞是来源于肝癌细胞，Huh7细胞能稳定产生IFN-β，并不适合作为HCV体外培养细胞，所以Blight等人在2003年用IFN-β预处理了含有Con1复制子的Huh7的细胞，得到的存活的细胞，命名为Huh7.5细胞[3]。由于Huh 7.5 RIG-I通道失活，不能产生大量的IFN-β，这些细胞可更高效的支持HCV复制[2, 3]。
　　2011年，Li等人建立了J6/JFH1重组体，该重组体是用HCV基因型2a型的J6的5’UTR-NS2替换了HCV基因型2a型的JFH1的5’UTR-NS2。该重组体能在Huh7.5细胞中产生的病毒适用于HCV的疫苗等研究[4]，该研究是HCV体外细胞培养的重大突破。
　　自1989年Choo等分离得到第一个HCV cDNA克隆以后，关于HCV的研究一直发展缓慢，直到1999年Lohmann等人第一次提出了复制子系统，HCV才有了第一个体外复制系统，该系统能高效的产生大量HCV蛋白，对于HCV体外培育提供了巨大的贡献及研究工具.对于HCV的疫苗和药物的研究仍有限制性。自Li等人于2010年用HCV基因型2a型的J6的5’UTR-NS2替换了HCV基因2a型的JFH1的5’UTR-NS2后，得到了能在Huh7.5细胞中稳定产生高滴度的重组HCV病毒[4]，该病毒能产生大量的具备HCV特征的蛋白及活病毒颗粒，得到的活病毒可以用于感染新的健康的Huh7.5细胞。这使得HCV的研究进入了新的研究领域。实现了体外培育HCV，并产生高滴度的HCV病毒，得到的病毒可以用于进行药物及疫苗的研究。
　　Huh7.5细胞来源于Huh7细胞，是Blight等人在2003年用IFN-β预处理了含有Con1复制子的Huh7的细胞，得到的存活的细胞，命名为Huh7.5细胞。研究发现Huh7.5细胞RIG-I通道失活，导致Huh7.5细胞IFN产生较Huh7细胞明显减少，适合作为HCV病毒体外培育的细胞[3]。因Huh7细胞因能产生较多IFN，不是最合适体外培育HCV病毒，因此Li的关于HCV的研究一直基于Huh7.5细胞基础上进行，在Huh7细胞基础上进行的研究较少。
　　本实验通过在Huh7细胞中培育新型HCV感染克隆，得到了带有新的突变的HCV病毒。并通过感染新的健康的Huh7细胞证明该病毒能产生感染性颗粒。
　　J6/JFH1重组病毒在转染至Huh7细胞时期，通过荧光显微镜观察到HCV阳性细胞量一直较低，直到第9天HCV阳性细胞才达到10%左右，此后HCV阳性细胞率逐渐上升，在第13天达到了80%左右，可能由于突变的病毒率较低，HCV阳性细胞在第15天迅速降至60%左右。
　　我们选取了转染时期第13天的病毒上清液感染新的Huh7细胞，得到的病毒称为第一代病毒。第一代病毒在感染Huh7细胞后的阳性率则能在第5日达到40%，这可能是由于第一代感染产生的HCV病毒在短时期内能在Huh7细胞内能达到稳定状态，并能迅速的感染及复制，该现象可能是由于该HCV病毒的基因序列发生了突变造成，该突变可能使该HCV病毒具有一定程度的抗IFN作用。
　　然后我们又用收集的一代病毒感染新的Huh7细胞，收集的病毒命名为二代病毒。在第三天观察感染效率，HCV阳性细胞达到了60%，第五天达到了80%，该时间小于第一次感染和转染时的时间，可能由于该HCV病毒在Huh7细胞中达到比一代病毒和二代病毒更为稳定的状态，所以在短时期内能迅速感染大量的Huh7细胞。通过病毒达到高峰的时间可以得出该病毒并不同于J6/JFH1。
　　通过测定转染、一代病毒和二代病毒的滴度，可以发现转染时期HCV阳性细胞转染效率达到80%时期的病毒滴度仍低于相同转染率在Huh7.5细胞内HCV阳性细胞转染率达到80%时期的病毒滴度，这可能是由于转染至Huh7细胞内的病毒仍不稳定，也可能是由于产生突变的病毒量较少所致。第二次感染收集的HCV病毒的滴度已经接近了相同转染率的Huh7.5细胞内产生HCV病毒的滴度。这可能是由于该HCV病毒已经在Huh7细胞内达到了相对稳定的状态并能产生大量感染性病毒颗粒所致。
　　本实验得到了不同于J6/JFH1病毒的新型丙肝感染性克隆，我们命名为J6/JFH1Huh7。可能是由于病毒带有了新型的适应性突变，该突变可能能使HCV病毒能在Huh7细胞内迅速达到稳定状态并能短期内产生大量的感染性病毒颗粒，由于后续的测序实验还在进行中，目前暂不清楚该适应性突变的位点及突变类型。
　　长期以来，丙肝病毒对于干扰素及利巴韦林的耐药率逐年增加，该实验成功建立了新型的感染细胞系，为耐IFN的丙肝治疗奠定了基础，为以后丙肝的治疗提供了新的方向。
　　【参考文献】
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