title: Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system.
abstract: 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.
authors.affiliation: Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kato
authors.forename: Tomoko
authors.initials: T
authors.lastname: Date
authors.forename: Michiko
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Masaya
authors.initials: M
authors.lastname: Sugiyama
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Etsuro
authors.initials: E
authors.lastname: Orito
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Ohno
authors.forename: Kanji
authors.initials: K
authors.lastname: Sugihara
authors.forename: Izumi
authors.initials: I
authors.lastname: Hasegawa
authors.forename: Kei
authors.initials: K
authors.lastname: Fujiwara
authors.forename: Kiyoaki
authors.initials: K
authors.lastname: Ito
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ozasa
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004357:Drug Synergism; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins; D012093:Replicon / Q000187:drug effects; D012254:Ribavirin / Q000494:pharmacology*; D014162:Transfection; D014773:Virology / Q000379:methods; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins; D012254:Ribavirin