Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals
Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals
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使用新策略开发丙型肝炎病毒基因型 6a 临床分离株的感染细胞培养系统及其对直接作用抗病毒药物的敏感性
DOI：
10.3389/fmicb.2018.02950
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发表时间：
2018-12-04
影响因子：
5.2
通讯作者：
Li, Yi-Ping
中科院分区：
文献类型：
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作者：
Chen, Mingxiao;Zheng, Fuxiang;Li, Yi-Ping
丙型肝炎病毒（HCV）分为7个主要基因型，其中基因6型在亚洲普遍流行，因此需要一个反映基因6型分离株流行情况的反向遗传系统。在这里，我们使用一种新的策略为中国HCV 6a分离株（CH6a）开发了一个传染性克隆。我们从患者血清中确定了CH6a一致性序列，并使用与一致性序列同源性最高的重叠PCR产物衍生克隆组装了CH6a全长（CH6aFL） cDNA。CH6aFL在肝癌Huh7.5细胞中无感染性。接下来，我们构建了含有CH6a中的Core-NS5A或5'UTR-NS5A以及JFH1（基因型2a）中剩余序列的重组体，并对两者进行了先前鉴定的7个突变的工程改造。然而，在没有病毒传播的情况下，它们在Huh7.5细胞中复制效率低下。加入CH6a Core-NS2重组体的适应性突变，加上JFH1 5'UTR和NS3-3'UTR，增强了Core-NS5A重组体的生存能力，获得了复制增强突变。CH6a重组体的22个突变与JFH1 5'UTR和3'UTR （CH6aORF）的结合使病毒能够复制，并恢复了另外4个突变。将这4个突变添加到Huh7.5.1-VISI-mCherry和Huh7.5细胞中，我们获得了两个高效的含26个突变（26m）的重组体CH6aORF_26m和CH6aFL_26m（命名为“CH6acc”），释放了104.3-104.5个聚焦形成单位(FFU)/ml的HCV。七个新发现的突变对HCV的复制、组装和释放很重要。靶向NS3/4A、NS5A和NS5B的直接作用抗病毒药物对CH6aORF_26m病毒呈剂量依赖性和基因型依赖性抑制。CH6acc丰富了HCV培养系统的工具箱，这里应用的策略和突变将促进其他HCV分离株和相关病毒的培养发展。
Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5′UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5′UTR and NS3-3′UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5′UTR and 3′UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated “CH6acc”), releasing HCV of 104.3–104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.