An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain
An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain
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DOI：
10.1111/j.1872-034x.2007.00056.x
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发表时间：
2007-06-01
影响因子：
4.2
通讯作者：
Wakita, Takaji
中科院分区：
文献类型：
--
作者：
Date, Tomoko;Miyamoto, Michiko;Wakita, Takaji
目的：从暴发性肝炎患者中克隆丙型肝炎病毒JFH-1株。JFH-1亚基因组复制子和全长JFH-1 RNA在培养细胞中有效复制。在本研究中，构建了一个具有传染性、选择性的HCV复制子，内含全长JFH-1 cDNA。方法：利用新霉素耐药基因、EMCV IRES和野生型JFH-1 cDNA构建全基因组复制子。用体外合成的RNA转染Huh7细胞，再用G418培养。克隆独立的菌落以建立复制全长HCV复制子的细胞系。结果：各分离细胞系均检测到HCV RNA复制。HCV蛋白和HCV RNA分泌到培养基中，呈现相同的密度分布。有趣的是，复制子细胞的培养上清液对未成熟的Huh7细胞具有传染性。长期培养不影响复制子细胞中复制子RNA的复制，但降低了核心蛋白的分泌和培养上清的感染性。复制子病毒连续传代后获得的培养上清对Huh7细胞具有感染性。结论：利用含有全长基因型2a JFH-1 cDNA的HCV复制子建立了可选择性感染。该系统可能对HCV研究有用。
Aim: The hepatitis C virus (HCV) strain JFH-1 was cloned from a patient with fulminant hepatitis. A JFH-1 subgenomic replicon and full-length JFH-1 RNA efficiently replicate in cultured cells. In this study, an infectious, selectable HCV replicon containing full-length JFH-1 cDNA was constructed.Methods: The full-genome replicon was constructed using the neomycin-resistant gene, EMCV IRES and wild-type JFH-1 cDNA. Huh7 cells were transfected with RNA synthesized in vitro, and then cultured with G418. Independent colonies were cloned to establish cell lines that replicate the full-length HCV replicon.Results: HCV RNA replication was detected in each isolated cell line. HCV proteins and HCV RNA were secreted into culture medium, and exhibited identical density profiles. Interestingly, culture supernatants of the replicon cells were infectious for naive Huh7 cells. Long-term culture did not affect replication of replicon RNA in the replicon cells, but it reduced core protein secretion and infectivity of culture supernatant. Culture supernatant obtained after serial passage of replicon virus was infectious for Huh7 cells.Conclusion: Selectable infection was established using HCV replicon containing full-length genotype 2a JFH-1 cDNA. This system might be useful for HCV research.