Background & Aims

Chronic liver diseases caused by hepatitis C virus (HCV) genotype 6 are prevalent in Asia, and millions of people require treatment with direct-acting antiviral regimens, such as NS5A inhibitor velpatasvir combined with the NS5B polymerase inhibitor sofosbuvir. We developed infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance.

Methods

The consensus sequences of strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants. We studied the efficacy of NS5A and NS5B inhibitors in concentration-response assays. We examined the effects of long-term culture of Huh7.5 cells incubated with velpatasvir and sofosbuvir singly or combined following infection with passaged full-length HK2 or HK6a recombinant viruses. Resistance-associated substitutions (RAS) were identified by Sanger and next-generation sequencing, and their effects on viral fitness and in drug susceptibility were determined in reverse-genetic experiments.

Results

Adapted full-length HCV genotype 6a recombinants HK2cc and HK6acc had fast propagation kinetics and high infectivity titers. Compared with an HCV genotype 1a recombinant, HCV genotype 6a recombinants of strains HK2 and HK6a were equally sensitive to daclatasvir, elbasvir, velpatasvir, pibrentasvir, and sofosbuvir, but less sensitive to ledipasvir, ombitasvir, and dasabuvir. Long-term exposure of HCV genotype 6a-infected Huh7.5 cells with a combination of velpatasvir and sofosbuvir resulted in clearance of the virus, but the virus escaped the effects of single inhibitors via emergence of the RAS L31V in NS5A (conferring resistance to velpatasvir) and S282T in NS5B (conferring resistance to sofosbuvir). Engineered recombinant genotype 6a viruses with single RAS mediated resistance to velpatasvir or sofosbuvir. HCV genotype 6a viruses with RAS NS5A-L31V or NS5B-S282T were however, able to propagate and escape in Huh7.5 cells exposed to the combination of velpatasvir and sofosbuvir. Further, HCV genotype 6a with NS5A-L31V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor.

Conclusions

Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle for this important genotype. The combination of velpatasvir and sofosbuvir is required to block propagation of original HCV genotype 6a, which quickly becomes resistant to single inhibitors via the rapid emergence and persistence of RAS. These features of HCV genotype 6a could compromise treatment.

中文翻译：

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在稳健的感染性细胞培养模型中，HCV 基因 6a 型逃逸和耐药性 Velpatasvir、Pibrentasvir 和 Sofosbuvir

背景与目标

由丙型肝炎病毒 (HCV) 基因 6 型引起的慢性肝病在亚洲普遍存在，数百万人需要使用直接作用的抗病毒方案进行治疗，例如 NS5A 抑制剂维帕他韦联合 NS5B 聚合酶抑制剂索非布韦。我们开发了 HCV 基因型 6a 感染的感染性细胞培养模型，以研究这些抑制剂的作用和耐药性的发展。

方法

源自慢性HCV感染患者血清的菌株HK2（MG717925）和HK6a（MG717928）的共有序列通过逆转录聚合酶链反应扩增的基因组的Sanger测序确定。这些 6a 菌株的体外非感染性全长克隆随后在 Huh7.5 细胞中进行了改造，主要是通过使用在基于 JFH1 的 Core-NS5A 和 Core-NS5B 基因型 6a 重组体中鉴定的替换。我们研究了 NS5A 和 NS5B 抑制剂在浓度反应试验中的功效。我们检查了 Huh7.5 细胞在感染传代的全长 HK2 或 HK6a 重组病毒后单独或联合培养的 Huh7.5 细胞的影响。通过 Sanger 和二代测序鉴定出耐药性相关取代（RAS），

结果

适应的全长HCV基因型6a重组体HK2cc和HK6acc具有快速传播动力学和高感染滴度。与 HCV 基因 1a 型重组体相比，HK2 和 HK6a 株的 HCV 基因 6a 型重组体对 daclatasvir、elbasvir、velpatasvir、pibrentasvir 和 sofosbuvir 的敏感性相同，但对 ledipasvir、ombitasvir 和 dasabuvir 的敏感性较低。HCV 基因型 6a 感染的 Huh7.5 细胞与维帕他韦和索非布韦的组合长期暴露导致病毒清除，但病毒通过在 NS5A 中出现 RAS L31V 逃脱了单一抑制剂的作用（赋予对维帕他韦的抗性) 和 NS5B 中的 S282T（赋予对索非布韦的耐药性）。工程重组基因 6a 型病毒，具有单一 RAS 介导的对维帕他韦或索非布韦的耐药性。然而，具有 RAS NS5A-L31V 或 NS5B-S282T 的 HCV 基因型 6a 病毒能够在暴露于维帕他韦和索非布韦组合的 Huh7.5 细胞中繁殖和逃逸。此外，具有 NS5A-L31V 的 HCV 基因型 6a 能够在 pibrentasvir 存在下繁殖和逃逸，同时出现 NS5A-L28S，从而赋予对该抑制剂的高水平抗性。

结论

从患者中分离出的 HCV 基因型 6a 菌株可以适应在培养细胞中繁殖，从而可以研究这一重要基因型的完整生命周期。需要 velpatasvir 和 sofosbuvir 的组合来阻止原始 HCV 基因型 6a 的传播，该基因型 6a 通过 RAS 的快速出现和持续存在而迅速对单一抑制剂产生耐药性。HCV 基因型 6a 的这些特征可能会影响治疗。