Highly Efficient Infectious Cell Culture of Three Hepatitis C Virus Genotype 2b Strains and Sensitivity to Lead Protease, Nonstructural Protein 5A, and Polymerase Inhibitors
Highly Efficient Infectious Cell Culture of Three Hepatitis C Virus Genotype 2b Strains and Sensitivity to Lead Protease, Nonstructural Protein 5A, and Polymerase Inhibitors
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DOI：
10.1002/hep.26660
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发表时间：
2014-02-01
期刊：
影响因子：
13.5
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Ramirez, Santseharay;Li, Yi-Ping;Bukh, Jens
丙型肝炎病毒（HCV）是一种基因多样化的病毒，多种基因型表现出显着差异，特别是在药物敏感性方面。药物和疫苗的开发将受益于模仿完整病毒生命周期的高滴度 HCV 培养物，但此类系统仅适用于基因型 1a 和 2a。我们为流行病学上重要的基因型 2b 开发了高效的培养系统。通过使用 F1468L/A1676S/D3001G (LSG) 突变，使患者菌株 DH8 和 DH10 的全长分子克隆适应在 Huh7.5 细胞中的有效生长。对先前开发的 J8cc 原型 2b 重组体进行了进一步改编。 DH8 和 J8 的感染滴度 >4.5 log10 病灶形成单位/mL。一组确定的 DH8 突变具有交叉隔离适应潜力。将 DH10 多蛋白编码序列插入具有 J8 非翻译区的载体中的嵌合基因组是可行的。重要的是，我们成功地生成了 DH8、J8 和 DH10 病毒，其在先导直接作用抗病毒药物的靶向区域中具有真实序列。非结构蛋白 (NS)5B 抑制剂索磷布韦、美西他滨和 BI207127 对 1a（TN 菌株）、2a（JFH1 和 J6 菌株）和 2b 菌株具有活性，而 VX-222 和 filibuvir 仅抑制 1a。基因型 2b 菌株对七种主要蛋白酶抑制剂最不敏感，其中包括具有高整体效力的 MK-5172。 NS5A 抑制剂达卡他韦非常有效，但疗效受到 HCV 毒株的影响。结论：使用具有明确突变的共有克隆可以建立高效的HCV全长2b培养系统。主要蛋白酶和 NS5A 抑制剂以及聚合酶抑制剂索非布韦、美西他滨和 BI207127 对全长 1a、2a 和 2b 病毒表现出交叉活性，但在分离株水平上存在重要的敏感性差异。不同HCV毒株的感染性培养将推进病毒生物学和发病机制的研究，并促进个体化患者治疗。 （肝病学 2014；59：395-407）
Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. Conclusion: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment. (Hepatology 2014;59:395-407)