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Journal of Clinical Microbiology, November 2005, p. 5679-5684, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5679-5684.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Detection of Anti-Hepatitis C Virus Effects of Interferon and Ribavirin by a Sensitive Replicon System

Takanobu Kato,1,2 Tomoko Date,2 Michiko Miyamoto,2 Masaya Sugiyama,1 Yasuhito Tanaka,1 Etsuro Orito,3 Tomoyoshi Ohno,3 Kanji Sugihara,3 Izumi Hasegawa,3 Kei Fujiwara,3 Kiyoaki Ito,3 Atsushi Ozasa,3 Masashi Mizokami,1 and Takaji Wakita2*

Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya,1 Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo,2 Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan3

Received 8 July 2005/ Returned for modification 17 August 2005/ Accepted 31 August 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 µg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 µg/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.

* Corresponding author. Mailing address: Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Phone: 81-423-25-3881. Fax: 81-423-21-8678. E-mail: wakita{at}tmin.ac.jp.

Journal of Clinical Microbiology, November 2005, p. 5679-5684, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5679-5684.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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