title: High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.
abstract: The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.
authors.affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wichroski
authors.forename: Jie
authors.initials: J
authors.lastname: Fang
authors.forename: Betsy J
authors.initials: BJ
authors.lastname: Eggers
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Rose
authors.forename: Charles E
authors.initials: CE
authors.lastname: Mazzucco
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Pokornowski
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Baldick
authors.forename: Monique N
authors.initials: MN
authors.lastname: Anthony
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Dowling
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Barber
authors.forename: John E
authors.initials: JE
authors.lastname: Leet
authors.forename: Brett R
authors.initials: BR
authors.lastname: Beno
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Gerritz
authors.forename: Michele L
authors.initials: ML
authors.lastname: Agler
authors.forename: Mark I
authors.initials: MI
authors.lastname: Cockett
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Tenney
mesh_terms: D000998:Antiviral Agents / Q000032:analysis* / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000187:drug effects; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents