Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Gastroenterology. 2018 Jun;154(8):2194-2208.e12. doi: 10.1053/j.gastro.2018.02.017. Epub 2018 Feb 15.
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.
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.
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.
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.
由丙型肝炎病毒（HCV）基因型 6 引起的慢性肝病在亚洲很常见，数以百万计的人需要用直接作用抗病毒药物治疗，如 NS5A 抑制剂伏西瑞韦联合 NS5B 聚合酶抑制剂索磷布韦。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。
通过逆转录聚合酶链反应扩增的基因组的 Sanger 测序确定了源自慢性 HCV 感染患者血清的株 HK2（MG717925）和 HK6a（MG717928）的共识序列。随后，通过在 Huh7.5 细胞中主要使用 JFH1 为基础的核心-NS5A 和核心-NS5B 基因型 6a 重组体中鉴定的取代来适应这些 6a 株的全长非感染性克隆。我们在浓度反应测定中研究了 NS5A 和 NS5B 抑制剂的功效。我们研究了长期培养 Huh7.5 细胞的效果，这些细胞在感染传代全长 HK2 或 HK6a 重组病毒后单独或联合使用伏西瑞韦和索磷布韦进行培养。通过 Sanger 和下一代测序鉴定耐药相关取代（RAS），并通过反向遗传实验确定它们对病毒适应性和药物敏感性的影响。
适应的全长 HCV 基因型 6a 重组体 HK2cc 和 HK6acc 具有快速繁殖动力学和高感染滴度。与 HCV 基因型 1a 重组体相比，株 HK2 和 HK6a 的 HCV 基因型 6a 重组体对达卡他韦、艾尔巴韦、伏西瑞韦、哌仑他韦和索磷布韦同样敏感，但对 ledipasvir、ombitasvir 和 dasabuvir 的敏感性较低。HCV 基因型 6a 感染的 Huh7.5 细胞长期暴露于伏西瑞韦和索磷布韦联合治疗可清除病毒，但病毒通过 NS5A 中的 RAS L31V（赋予对伏西瑞韦的耐药性）和 NS5B 中的 S282T（赋予对索磷布韦的耐药性）的出现而逃避了单一抑制剂的作用。工程重组基因型 6a 病毒具有对伏西瑞韦或索磷布韦的单一 RAS 介导的耐药性。然而，具有 NS5A-L31V 或 NS5B-S282T 的 HCV 基因型 6a 病毒能够在暴露于伏西瑞韦和索磷布韦联合治疗的 Huh7.5 细胞中繁殖并逃逸。此外，具有 NS5A-L31V 的 HCV 基因型 6a 能够在出现 NS5A-L28S 时繁殖并逃逸，这赋予了对该抑制剂的高耐药性。
从患者中分离的 HCV 基因型 6a 株可适应在培养细胞中繁殖，从而允许对这种重要基因型的完整生命周期进行研究。需要伏西瑞韦和索磷布韦联合阻断原始 HCV 基因型 6a 的繁殖，因为 RAS 的快速出现和持续存在会导致该病毒很快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效果。
