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High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

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.

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Related in: MedlinePlus

Optimization of a high-throughput 384 well virus replication assay.A. Comparison of virus replication over time (cells/viral foci) following infection using a standard protocol where virus was added to adherent Huh-7.5 cells (pre-plated 24 h prior to infection) or a homogenous protocol where virus and trypsinized cells were co-dispensed into a well. B. Co-titration of genotype 1a/2a-Rluc virus and Huh-7.5 cells to determine Z factor values in a 96 h assay. C. Effect of MOI on % inhibition obtained with entry (EI), genome replication (BMS-339) and virus assembly (LY411575) inhibitors in a 96 h assay. D. HTS assay strategy. E & F. HTS assay quality control. Plate-to-plate variability in signal/backgroundr (E) and Z factor (F) was determined for the Renilla luciferase and HCV Core Cellomics ArrayScan readouts from 50 and 12 plates validation runs, respectively. Results are expressed as mean and standard deviation.
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pone-0042609-g002: Optimization of a high-throughput 384 well virus replication assay.A. Comparison of virus replication over time (cells/viral foci) following infection using a standard protocol where virus was added to adherent Huh-7.5 cells (pre-plated 24 h prior to infection) or a homogenous protocol where virus and trypsinized cells were co-dispensed into a well. B. Co-titration of genotype 1a/2a-Rluc virus and Huh-7.5 cells to determine Z factor values in a 96 h assay. C. Effect of MOI on % inhibition obtained with entry (EI), genome replication (BMS-339) and virus assembly (LY411575) inhibitors in a 96 h assay. D. HTS assay strategy. E & F. HTS assay quality control. Plate-to-plate variability in signal/backgroundr (E) and Z factor (F) was determined for the Renilla luciferase and HCV Core Cellomics ArrayScan readouts from 50 and 12 plates validation runs, respectively. Results are expressed as mean and standard deviation.

Mentions: Rapid screening of large-scale compound libraries requires homogenous, miniaturized platforms that can be automated. A first step towards assay simplification was to determine if virus and trypsinized cells could be mixed in suspension to initiate infection, and dispensed onto plates containing screen compounds, instead of adding virus to adherent cells plated 24 hours prior. It was observed that the effective titer of a virus stock (ffu per well) was typically reduced approximately 1.5-fold in the one-step procedure relative to a multi-step infection protocol (data not shown) and the expansion of viral foci (virus per foci) was only modestly (<2 fold) delayed (Fig. 2A). Importantly, the homogenous protocol had no impact on the potency of control inhibitors (data not shown).


High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

Optimization of a high-throughput 384 well virus replication assay.A. Comparison of virus replication over time (cells/viral foci) following infection using a standard protocol where virus was added to adherent Huh-7.5 cells (pre-plated 24 h prior to infection) or a homogenous protocol where virus and trypsinized cells were co-dispensed into a well. B. Co-titration of genotype 1a/2a-Rluc virus and Huh-7.5 cells to determine Z factor values in a 96 h assay. C. Effect of MOI on % inhibition obtained with entry (EI), genome replication (BMS-339) and virus assembly (LY411575) inhibitors in a 96 h assay. D. HTS assay strategy. E & F. HTS assay quality control. Plate-to-plate variability in signal/backgroundr (E) and Z factor (F) was determined for the Renilla luciferase and HCV Core Cellomics ArrayScan readouts from 50 and 12 plates validation runs, respectively. Results are expressed as mean and standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g002: Optimization of a high-throughput 384 well virus replication assay.A. Comparison of virus replication over time (cells/viral foci) following infection using a standard protocol where virus was added to adherent Huh-7.5 cells (pre-plated 24 h prior to infection) or a homogenous protocol where virus and trypsinized cells were co-dispensed into a well. B. Co-titration of genotype 1a/2a-Rluc virus and Huh-7.5 cells to determine Z factor values in a 96 h assay. C. Effect of MOI on % inhibition obtained with entry (EI), genome replication (BMS-339) and virus assembly (LY411575) inhibitors in a 96 h assay. D. HTS assay strategy. E & F. HTS assay quality control. Plate-to-plate variability in signal/backgroundr (E) and Z factor (F) was determined for the Renilla luciferase and HCV Core Cellomics ArrayScan readouts from 50 and 12 plates validation runs, respectively. Results are expressed as mean and standard deviation.
Mentions: Rapid screening of large-scale compound libraries requires homogenous, miniaturized platforms that can be automated. A first step towards assay simplification was to determine if virus and trypsinized cells could be mixed in suspension to initiate infection, and dispensed onto plates containing screen compounds, instead of adding virus to adherent cells plated 24 hours prior. It was observed that the effective titer of a virus stock (ffu per well) was typically reduced approximately 1.5-fold in the one-step procedure relative to a multi-step infection protocol (data not shown) and the expansion of viral foci (virus per foci) was only modestly (<2 fold) delayed (Fig. 2A). Importantly, the homogenous protocol had no impact on the potency of control inhibitors (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

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.

Show MeSH
Related in: MedlinePlus