Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses
Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses
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DOI：
10.1053/j.gastro.2007.08.005
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发表时间：
2007-11-01
期刊：
影响因子：
29.4
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Gottwein, Judith M.;Scheel, Troels K. H.;Bukh, Jens
背景和目的：最近，针对JFH1株（基因型2a）和基因内型2a/2a基因组（J6/JFH）开发了完整的病毒生命周期丙型肝炎病毒（HCV）细胞培养系统。我们的目标是利用独特的 JFH1 复制特征来开发基因型 3a 的培养系统，该基因型在全球范围内流行率很高。方法：用具有 3a 参考毒株 S52 核心、E1、E2、p7 和 NS2 的基因间 3a/JFH1 重组体的 RNA 转录物转染 Huh7.5 细胞，并传代释放的病毒。检查培养物的 HCV 核心和/或 NS5A 表达（免疫染色）、HCV RNA 滴度（实时 PCR）和感染滴度（50% 组织培养感染剂量）。通过反向遗传学研究分析了通过对回收的 S52/JFH1 病毒进行测序鉴定的突变的作用。结果：在 Huh7.5 细胞中传代的 S52/JFH1 和 J6/JFH 病毒显示出相当的生长动力学和相似的 HCV RNA 峰值和感染滴度。然而，对 S52/JFH1 病毒的分析在 core、E2、p7、NS3 和 NSSA 中发现了 9 个假定的适应性突变。所有 7 个 S52/JFH1 重组体在 p7 中发生氨基酸变化并结合 NS3 或 NS5A 中的变化，但 9 个具有单独突变（分别在 p7 和 NS3 中）的重组体中只有 2 个完全可行，不需要额外的突变。通过研究 3a/JFH1 感染对 CD81 的依赖性及其对细胞内脂质分布的影响，显示了我们系统的生物学相关性。结论：我们开发了一种针对 HCV 基因型 3a 的强大的基因型间重组细胞培养系统，为 3a core-NS2 和相关治疗的研究提供了有价值的工具。
Background & Aims: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide. Methods: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/ JFH1 viruses was analyzed by reverse genetics studies. Results: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NSSA. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids. Conclusions: We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.