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J Virol:我国首例丙型肝炎病毒感染性DNA克隆

来源:上海巴斯德研究所 2013-11-21 18:38

PR63cc病毒的特性研究。(A)PR63cc可感染Huh7.5.1细胞,蓝色为核,红色为HCV E2蛋白。(B)JFH-1PR63cc均可在Huh7.5.1细胞中高效扩增。(C)HCV NS5A抑制剂DaclatasvirJFH-1PR63ccPR63cc-NS5A-M31L的抑制曲线。

11月13日,国际学术期刊Journal of Virology在线发表了中科院上海巴斯德研究所钟劲课题组的研究论文A novel strategy to develop robust infectious hepatitis C virus cell culture system directly from a clinical isolate (《从丙型肝炎病毒临床分离株直接建立高效的丙型肝炎病毒细胞感染模型的新型策略》)。

丙型肝炎病毒(hepatitis C virus, HCV)感染是慢性肝病的主要致病因素之一,可诱发肝炎、肝硬化和肝癌。HCV的细胞感染模型对于HCV的生物学研究、抗病毒药物和疫苗的研发具有非常重要的作用,其中HCV感染性cDNA克隆的建立非常关键。2005年,基于JFH-1感染性克隆的HCV细胞培养模型的成功构建,极大地推动了HCV研究领域的发展。由于HCV基因组的变异很大,不同基因型HCV对治疗方案的敏感性有所不同,而且病毒基因型对致病性也有影响,因此很有必要建立更多的来源于不同感染性克隆的细胞感染模型。

本论文的第一作者、博士研究生卢捷和实验室其他研究人员合作,利用一例基因型2a HCV临床分离株(PR63)样本,通过功能性克隆筛选、拼接及引入细胞适应性突变,构建了它的全长感染性cDNA克隆(PR63cc),进而建立了基于PR63cc的HCV细胞感染模型。PR63cc在Huh7.5.1细胞中能高效产生病毒,但其对现有抗丙肝药物的敏感性和JFH-1病毒不同,其中PR63cc病毒对NS5A的抑制剂高度不敏感。

PR63cc是第一例来源于中国丙肝患者样本的全长感染性克隆,也是世界上首例不通过共有序列,而直接从临床样本来源构建的能感染肝细胞系的感染性克隆。这种新开发的方法可以应用于其它临床分离株,对丙型肝炎患者的个性化治疗、药物测试开发等领域均有重要意义。重要的是,PR63cc病毒株的构建方法及毒株本身已经申请专利,具有自主知识产权,为下一步抗HCV药物和疫苗的研发带来极大便利。

该项研究工作是和上海交通大学附属瑞金医院谢青教授课题组合作完成,得到了科技部“973”、传染病重大科技专项等经费的支持。(生物谷Bioon.com)

生物谷推荐的英文摘要

Journal of Virology             doi: 10.1128/JVI.02929-13

A novel strategy to develop robust infectious hepatitis C virus cell culture system directly from a clinical isolate

Jie Lu1, Yu Xiang1, Wanyin Tao1, Qingchao Li1, Na Wang1, Yongfeng Gao1, Xiaogang Xiang2, Qing Xie2 and Jin Zhong1

HCV infection is a leading cause of chronic liver diseases. The progress in HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and inter-genotypic recombinants were developed and allowed the study of vaccines and antiviral inhibitors for all genotypes. Only until recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. In this study, we developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, which could efficiently produce virus particles in Huh7 derived cells, with peak titers of 1.6×105 ffu/mL. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents, but appeared more resistant to an NS5A inhibitor than JFH-1. In summary: we developed a new approach to construct infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.

 

 

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