Division of Viral Products, Center for Biologics Evaluation and Research, FDA, 29 Lincoln Drive, Bethesda, MD 20892, USA.
Virol J. 2011 Aug 15;8:402. doi: 10.1186/1743-422X-8-402.
Given the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recently published clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Therefore, understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro.
We serially passed the J6/JFH1 strain of HCV in Huh7D cells (a Huh7 cell derivative more permissive to HCV replication) in the presence of different concentrations of ribavirin. Virus replication was assessed by detection of HCV antigens by immunfluorscence of infected cells and titration of recovered virus present in the supernatant. cDNAs from virus RNA grown in 0 or 250 uM concentrations of ribavirin were synthesized by RT-PCR, and sequenced.
A concentration of 125 uM of ribavirin did not have a dramatic effect on HCV replication, while 500 uM of ribavirin lead to viral extinction. Concentrations of 250 uM of ribavirin dramatically reduced virus replication which was sustained over six passages. At passage seven viral resurgence began and over two passages the level of virus reached that of the wild type virus grown without ribavirin. Virus recovered from these cultures were more resistant to 250 uM ribavirin than wild type virus, and showed no difference in replication relative to wild type virus when grown in the absence of ribavirin. The ribavirin resistant virus accumulated multiple synonymous and non-synonymous mutations that are presently being analyzed for their relationship to ribavirin resistance.
It is possible to select a ribavirin resistant mutant of HCV that can replicate to levels similar to wild type virus grown without ribavirin. Analysis of the mutations responsible for the ribavirin resistance may aid in understanding the mechanism of action of ribavirin.
鉴于与干扰素静脉注射相关的副作用，人们非常希望有一种治疗 HCV 感染的无干扰素方案。最近发表的临床研究表明，含有利巴韦林的无干扰素联合疗法是有效的，这表明无干扰素治疗可能在不久的将来被采用。因此，了解 HCV 对利巴韦林的耐药性可能非常重要。为了了解利巴韦林对 HCV 复制和 HCV 耐药性的影响，我们在体外选择了一株 HCV 利巴韦林耐药突变株。
我们在不同浓度利巴韦林存在的情况下，将 J6/JFH1 株 HCV 在 Huh7D 细胞（一种对 HCV 复制更具包容性的 Huh7 细胞衍生物）中连续传代。通过感染细胞的免疫荧光检测和上清液中回收病毒的滴定来评估病毒复制。在 0 或 250 uM 利巴韦林浓度下生长的病毒 RNA 的 cDNA 通过 RT-PCR 合成，并进行测序。
125 uM 利巴韦林的浓度对 HCV 复制没有显著影响，而 500 uM 利巴韦林则导致病毒灭绝。250 uM 利巴韦林的浓度显著降低了病毒复制，这种情况持续了六代。在第七代时病毒开始反弹，经过两代后，病毒水平达到了无利巴韦林生长的野生型病毒水平。从这些培养物中回收的病毒对 250 uM 利巴韦林的耐药性比野生型病毒更高，并且在无利巴韦林生长时与野生型病毒的复制没有差异。耐药病毒积累了多个同义和非同义突变，目前正在分析这些突变与利巴韦林耐药性的关系。
可以选择一株能够复制到与无利巴韦林生长的野生型病毒相似水平的利巴韦林耐药 HCV 突变株。对导致利巴韦林耐药的突变的分析可能有助于了解利巴韦林的作用机制。
