Kato Takanobu, Date Tomoko, Miyamoto Michiko, Sugiyama Masaya, Tanaka Yasuhito, Orito Etsuro, Ohno Tomoyoshi, Sugihara Kanji, Hasegawa Izumi, Fujiwara Kei, Ito Kiyoaki, Ozasa Atsushi, Mizokami Masashi, Wakita Takaji
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.
J Clin Microbiol. 2005 Nov;43(11):5679-84. doi: 10.1128/JCM.43.11.5679-5684.2005.
Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 microg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 microg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.
虽然干扰素和利巴韦林联合疗法改善了慢性丙型肝炎病毒(HCV)感染的治疗,但利巴韦林在临床浓度下的详细抗HCV效果仍不确定。为检测较低浓度下利巴韦林的抗HCV效果,利用带有HCV 2a基因型亚基因组复制子(克隆JFH - 1)的报告基因复制子系统开发了一种灵敏且准确的检测系统,该复制子在多种细胞系中能强劲复制。这个报告基因复制子是通过将荧光素酶报告基因(而非新霉素抗性基因)导入亚基因组JFH - 1复制子而产生的。为评估该报告基因复制子的复制情况,在瞬时转染Huh7细胞后连续3天测量荧光素酶活性。在实验过程中,荧光素酶活性呈指数增长。在调整转染效率和转染细胞活力后,确定了干扰素和利巴韦林的影响。干扰素和利巴韦林的给药导致复制子RNA复制呈剂量依赖性抑制。干扰素和利巴韦林的50%抑制浓度分别为1.80 IU/ml和3.7 μg/ml。在临床浓度下,干扰素(100 IU/ml)和利巴韦林(3 μg/ml)分别将复制降低至0.09%和53.74%。利巴韦林与干扰素联合使用使干扰素的抗HCV效果增强了1.46至1.62倍。总之,我们开发了一种准确且灵敏的复制子系统,通过该复制子系统能够在干扰素和利巴韦林的临床浓度范围内轻松检测到它们的抗病毒效果。该系统将为筛选抗HCV的新型抗病毒化合物提供有力工具。