Division of Viral Products, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, United States of America.
PLoS One. 2013 Sep 5;8(9):e74027. doi: 10.1371/journal.pone.0074027. eCollection 2013.
While ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are also efficacious, suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross resistance to other drugs could impair the efficacy of the treatment. Therefore, understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance.
We tested the ability of a J6/JFH1 derived HCV ribavirin resistant mutant to grow in tissue cultured Huh7D cells in the presence of the mutagen 5-Fluorouracil and the nucleoside analog 2'-C-Methylcytidine. Virus replication was assessed by detecting HCV antigens by immunofluorescence and by titrating virus present in the supernatants. Recovered viruses were amplified by RT-PCR and sequenced.
The sensitivity of HCV-RR relative to parental J6/JFH1 to the tested drugs varied. HCV-RR was more resistant than J6/JFH1 to 5-Fluorouracil but was not more resistant than J6/JFH1 to 2'-C-Methylcytidine. Growth of HCV-RR in 5-Fluorouracil allowed the selection of an HCV-RR derived mutant resistant to 5-Fluorouracil (HCV-5FU). HCV-5FU grows to moderate levels in the presence of high concentrations of 5-Fluorouracil and to parental levels in the absence of the drug. Sequence of its genome shows that HCV-5FU accumulated multiple synonymous and non-synonymous mutations.
These results indicate that determinants of resistance to ribavirin could also confer resistance to other anti-HCV drugs, shedding light toward understanding the mechanism of action of ribavirin and highlighting the importance of combination drug selection for HCV treatment. The results also show that it is possible to select a 5-Fluorouracil HCV resistant mutant that replicates to levels similar to parental virus when grown in the absence of 5-Fluorouracil.
虽然利巴韦林单药治疗方案对慢性丙型肝炎病毒（HCV）感染患者的疗效甚微，但与干扰素联合使用时可能有效。临床研究表明，含有利巴韦林的无干扰素联合治疗也是有效的，这表明无干扰素治疗可能在不久的将来被采用。然而，耐药突变体的产生和对其他药物的交叉耐药性可能会影响治疗效果。因此，了解 HCV 对利巴韦林的耐药机制和对其他抗病毒药物的交叉耐药性可能非常重要。
我们测试了一种源自 J6/JFH1 的 HCV 利巴韦林耐药突变体在 5-氟尿嘧啶和核苷类似物 2'-C-甲基胞苷存在的情况下在组织培养的 Huh7D 细胞中生长的能力。通过免疫荧光法检测 HCV 抗原和测定上清液中存在的病毒来评估病毒复制。通过 RT-PCR 扩增和测序回收病毒。
与亲本 J6/JFH1 相比，HCV-RR 对测试药物的敏感性不同。HCV-RR 对 5-氟尿嘧啶的耐药性强于 J6/JFH1，但对 2'-C-甲基胞苷的耐药性不如 J6/JFH1。HCV-RR 在 5-氟尿嘧啶中的生长允许选择对 5-氟尿嘧啶耐药的 HCV-RR 衍生突变体（HCV-5FU）。HCV-5FU 在高浓度 5-氟尿嘧啶存在下生长到中等水平，在没有药物的情况下生长到亲本水平。其基因组序列表明，HCV-5FU 积累了多个同义和非同义突变。
这些结果表明，对利巴韦林的耐药决定因素也可能导致对其他抗 HCV 药物的耐药性，这为理解利巴韦林的作用机制提供了线索，并强调了联合药物选择对 HCV 治疗的重要性。结果还表明，有可能选择一种在没有 5-氟尿嘧啶的情况下复制到与亲本病毒相似水平的 5-氟尿嘧啶 HCV 耐药突变体。
