Hepatitis C Virus: Propagation, Quantification, and Storage

MinKyung Yi1

1 The University of Texas Medical Branch at Galveston, Galveston, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15D.1
DOI:  10.1002/9780471729259.mc15d01s19
Online Posting Date:  November, 2010
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Abstract

Hepatitis C virus (HCV) is a leading cause of chronic liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is well known for its restricted tropism and it does not replicate well in animal species other than humans and chimpanzees. Since classical in vitro propagation of natural HCV isolates is not possible, a protocol for the rescue of infectious virus from cDNA clones (genotype 1a pH77S and genotype 2a pJFH‐1) transfected as RNA into permissive cells is described here. Because these two molecular clones behave differently in their ability to propagate and produce infectious virus, different methods for propagation of these two viral strains are described. Methods for infectious virus titration, which can be accomplished by counting foci of infected cells following immunostaining for viral antigen expression in cells infected with serial dilutions of a virus harvest (focus forming unit, or FFU, assay), are also provided. Curr. Protoc. Microbiol. 19:15D.1.1‐15D.1.11. © 2010 by John Wiley & Sons, Inc.

Keywords: HCV; H77S; JFH‐1; immunostaining; FFU

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Virus from Cell Culture Infectious cDNA Clones
  • Basic Protocol 2: Propagation of Genotype 1a H77S and Its Derivatives
  • Basic Protocol 3: Propagation of Genotype 2a JFH‐1 and Its Derivatives
  • Basic Protocol 4: Titration of HCV
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Generation of Virus from Cell Culture Infectious cDNA Clones

  Materials
  • Huh‐7 cell line and its derivatives (e.g., S5C‐500‐3, 8C1, FT3‐7, Huh7.5)
  • Phosphate buffered saline (PBS; appendix 2A), ice‐cold
  • Trypsin
  • In vitro‐transcribed, full‐length H77S or JFH‐1‐derived RNA
  • Complete medium: DMEM with 10% FBS and other supplements (see recipe)
  • Fetal bovine serum (FBS)
  • 0.4‐cm gap‐width electroporation cuvette
  • Bio‐Rad Gene Pulser Xcell electroporation apparatus or equivalent
  • 25‐cm2 flasks
  • 37°C, 5% CO 2 humidified incubator
  • Centrifuge
  • 0.2‐µm PES (polyethersulfone) filters, optional
  • Additional reagents and equipment for culture of Huh‐7 cell lines [see Phelan ( ) for general cell culture methods] and performing electroporation (see Potter, )

Basic Protocol 2: Propagation of Genotype 1a H77S and Its Derivatives

  Materials
  • In vitro‐transcribed, full‐length H77S or its derivative RNA
  • Huh‐7 cell line and its derivatives (e.g., S5C‐500‐3, 8C1, Huh7.5)
  • Phosphate buffered saline (PBS; appendix 2A), ice cold
  • Complete medium: DMEM with 10% FBS and other supplements (see recipe)
  • Serum (FBS)‐free medium
  • Fetal bovine serum (FBS)
  • Electroporation cuvettes
  • Bio‐Rad Gene Pulser Xcell electroporation apparatus or equivalent
  • 175‐cm2 flasks
  • 37°C, 5% CO 2 humidified incubator
  • Centrifuge
  • Centricon Plus‐20 (UFC2BHK08), Centricon Plus‐70 (UFC710008) (Millipore)

Basic Protocol 3: Propagation of Genotype 2a JFH‐1 and Its Derivatives

  Materials
  • Huh‐7 cell line or its derivatives (e.g., FT3‐7, Huh7.5)
  • JFH‐1‐derived virus stock (see protocol 1) frozen or stored at 4°C
  • Complete medium: DMEM with 10% FBS and other supplements (see recipe)
  • Serum‐free medium or low‐percentage serum medium
  • Fetal bovine serum (FBS)
  • 25‐cm2 flasks
  • 37°C water bath
  • 37°C incubator/37°C, 5% CO 2 humidified incubator
  • Centrifuge
  • 0.2‐µm PES filter, optional
  • Centricon Plus‐20 (UFC2BHK08), Centricon Plus‐70 (UFC710008) (Millipore)

Basic Protocol 4: Titration of HCV

  Materials
  • Huh‐7.5 or S5C‐500‐3 cells in culture
  • Virus inoculum
  • Complete medium (see recipe)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1:1 (v/v) methanol/acetone solution (see recipe)
  • Mouse monoclonal anti‐core antibody (Affinity Bioreagent, C7‐50, or any other HCV antibodies suitable for immunostaining of HCV antigen)
  • Bovine serum albumin (7.5% BSA in DPBS, Sigma)
  • Alexa 488‐conjugated, goat‐anti‐mouse IgG antibody (Invitrogen)
  • Hoechst 33258, 10 mg/ml (Bisbenzimide)
  • VECTASHIELD mounting fluid (Vector Labs)
  • 8‐well tissue culture chamber slide
  • 37°C, 5% CO 2 humidified incubator
  • Coverslips
  • Epifluorescence microscope
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Literature Cited

Literature Cited
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