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      Cell‐Based Hepatitis C Virus Infection Fluorescence Resonance Energy Transfer (FRET) Assay for Antiviral Compound Screening

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          Abstract

          Hepatitis C virus (HCV) affects an estimated 3% of the population and is a leading cause of chronic liver disease worldwide. Since HCV therapeutic and preventative options are limited, the development of new HCV antivirals has become a global health care concern. This has spurred the development of cell‐based infectious HCV high‐throughput screening assays to test the ability of compounds to inhibit HCV infection. This unit describes methods that may be used to assess the in vitro efficacy of HCV antivirals using a cell‐based high‐throughput fluorescence resonance energy transfer (FRET) HCV infection screening assay, which allows for the identification of inhibitors that target HCV at any step in the viral life cycle. Basic protocols are provided for compound screening during HCV infection and analysis of compound efficacy using an HCV FRET assay. Support protocols are provided for propagation of infectious HCV and measurement of viral infectivity. Curr. Protoc. Microbiol. 18:17.5.1‐17.5.27. © 2010 by John Wiley & Sons, Inc.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          P. Chomczynski, N Sacchi (1987)
          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Robust hepatitis C virus infection in vitro.

            Jin Zhong, Pablo Gastaminza, Guofeng Cheng … (2005)
            The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.
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              Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.

              Mansun Law, Toshiaki Maruyama, Jamie Lewis … (2008)
              A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.
              Article 0 comments Cited 203 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Curr Protoc Microbiol
                Journal ID (iso-abbrev): Curr Protoc Microbiol
                Journal ID (doi): 10.1002/(ISSN)1934-8533
                Journal ID (publisher-id): CPMC
                Title: Current Protocols in Microbiology
                Publisher: John Wiley and Sons Inc. (Hoboken )
                ISSN (Print): 1934-8525
                ISSN (Electronic): 1934-8533
                Publication date (Electronic): 01 August 2010
                Publication date (Print): August 2010
                Volume: 18
                Issue: 1 , Anti‐Infectives ( doiID: 10.1002/9780471729259.2010.18.issue-1 )
                Pages: 1751-17527
                Affiliations
                [ 1 ] Department of Medicine, University of Illinois at Chicago Chicago Illinois
                [ 2 ] Department of Microbiology and Immunology, University of Illinois at Chicago Chicago Illinois
                Article
                Publisher ID: CPMC1705
                DOI: 10.1002/9780471729259.mc1705s18
                PMC ID: 2964379
                PubMed ID: 20812217
                SO-VID: 49da4b73-d859-467f-b93e-4a65e284388a
                Copyright © Copyright © 2010 John Wiley & Sons, Inc.
                License:

                This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

                History
                Page count
                Figures: 3, Tables: 2, Pages: 27, Words: 13523
                Categories
                Subject: Life Sciences Special Topics
                Subject: Microbiology
                Subject: Anti‐Infectives
                Subject: RNA Viruses
                Subject: Pharmacology and Drug Discovery
                Subject: Drug Discovery Technologies
                Subject: Anti‐Infectives
                Subject: Intermolecular Interactions
                Subject: Fret
                Subject: Unit
                Subject: Unit
                Custom metadata
                source-schema-version-number 2.0
                cover-date August 2010
                details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:15.04.2020

                Keywords: hepatitis c virus,viral lifecycle,huh7 cells,dimethylsulfoxide (dmso),fluorescence resonance energy transfer (fret),ns3 protease,antivirals,high‐throughput screening
                Data availability:
                Keywords: hepatitis c virus, viral lifecycle, huh7 cells, dimethylsulfoxide (dmso), fluorescence resonance energy transfer (fret), ns3 protease, antivirals, high‐throughput screening

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