Propagation, Quantification, Detection, and Storage of West Nile Virus

James D. Brien1, Helen M. Lazear1, Michael S. Diamond2

1 These authors contributed equally to this work., 2 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15D.3
DOI:  10.1002/9780471729259.mc15d03s31
Online Posting Date:  November, 2013
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Abstract

West Nile virus (WNV) is a member of the Flaviviridae family of enveloped, single‐stranded, positive‐sense RNA viruses. WNV, an emerging viral pathogen, is transmitted by mosquitoes to birds and mammals and is responsible for an increasing incidence of human disease in North America and Europe. Due to its ease of use in the laboratory and the availability of robust mouse models of disease, WNV provides an excellent experimental system for studying molecular virology and pathogenesis of infection by flaviviruses. Here, we describe common laboratory techniques used to propagate, quantify, detect, and store WNV. We also briefly describe appropriate safety precautions required for the laboratory use of WNV, which is classified as a Biosafety Level 3 pathogen by the United States Centers for Disease Control and Prevention. Curr. Protoc. Microbiol. 31:15D.3.1‐15D.3.18. ©2013 by John Wiley & Sons, Inc.

Keywords: West Nile virus; infection; titration; plaque assay; purification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and Purification of West Nile Virus Stocks
  • Basic Protocol 2: Quantification of WNV by Plaque Assay
  • Basic Protocol 3: Determination of WNV RNA by Quantitative Fluorogenic Reverse‐Transcription PCR
  • Basic Protocol 4: Viral Growth Curves
  • Alternate Protocol 1: Quantification of WNV by Focus‐Forming Assay
  • Support Protocol 1: Culturing BHK21 and Vero cells for WNV Propagation and Titering
  • Support Protocol 2: Propagation of C6/36 Insect Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation and Purification of West Nile Virus Stocks

  Materials
  • Vero cells ( protocol 6) or C6/36 cells ( protocol 7)
  • Medium for chosen cell type (see reciperecipes)
  • WNV New York 1999 seed stock (BEI Resources; http://www.beiresources.org)
  • Dulbecco's modified Eagle medium (DMEM, high‐glucose) containing 2% fetal bovine serum (FBS)
  • 25% glycerol in TNE (v/v) (see recipe for TNE)
  • TNE (see recipe)
  • T150 tissue culture flasks
  • 50‐ml disposable polystyrene conical tubes with screw caps (e.g., Falcon)
  • 1.5‐ml or 0.5‐ml O‐ring tubes
  • Beckman Coulter Optima L‐100 biosafe ultracentrifuge and rotor (e.g., SW32)
  • Beckman Coulter Polyallomer ultracentrifuge tubes
  • Sonicator (Branson Sonifier Sonicator with Cup‐Horn attachment) or glass Dounce homogenizer (optional)
  • Additional reagents and equipment for quantification of WNV by plaque assay ( protocol 2) or focus‐forming assay ( protocol 5)

Basic Protocol 2: Quantification of WNV by Plaque Assay

  Materials
  • BHK21 clone 15 cells ( protocol 6) and medium (see recipe)
  • 2× MEM (see recipe) + 8% FBS
  • Virus to be titered (from protocol 1 or protocol 4)
  • DMEM + 2% FBS
  • Low‐melt agarose (see recipe)
  • 10% formaldehyde
  • Crystal violet staining solution (see recipe)
  • 6‐well tissue culture plates
  • Water bath preset to 56°C
  • 96‐well U‐bottom plates
  • Multichannel pipettor
  • Styrofoam rack or other non‐conductive surface
  • Light box

Basic Protocol 3: Determination of WNV RNA by Quantitative Fluorogenic Reverse‐Transcription PCR

  Materials
  • Primer sets (forward, reverse, probe)
    • WNV NY‐1999 (Lineage I) (Lazear et al., ):
      • Fwd: 5′‐TCA GCG ATC TCT CCA CCA AAG‐3′
      • Rev: 5′‐GGG TCA GCA CGT TTG TCA TTG‐3′
      • Probe: 5′‐/56‐FAM/TGC CCG ACC ATG GGA GAA GCT C/36‐TAMSp/‐3′
    • WNV Madagascar 78 (AnMg798 , Lineage II) (Lazear et al., ):
      • Fwd: 5′‐TCA GTG AGT TAT CAA CAA GAG‐3′
      • Rev: 5′‐ GGA TCA GCT CTT TTC TCA TTA‐3′
      • Probe: 5′‐/56‐FAM/TGC CCA ACC ATG GGA GAA GCC C/36‐TAMSp/‐3′
    • WNV Kunjin (Daffis et al., ):
      • Fwd: 5′‐TCA GTG AAC CCA TCT CCA AGG‐3′
      • Rev: 5′‐GGG TCA GCC CGC TTG TCA TTA‐3′
      • Probe: 5′‐/56‐FAM/TGC CCA ACC ATG GGG GAA GCC C/36‐TAMSp/‐3′
  • Viral RNA experimental samples (store at −80°C)
  • Viral RNA standard (store at −80°C)
  • TaqMan One‐Step RT‐PCR Master Mix Reagents Kit (Applied Biosystems, cat. no. 4309169; store at 4°C)
    • 2× PCR mix
    • 40× MultiScribe reverse transcriptase
  • Nuclease‐free H 2O (use for all steps requiring H 2O)
  • MicroAmp Fast Optical 96‐well reaction plate (Applied Biosystems #4346906)
  • Optical adhesive cover (Applied Biosystems #4360954)
  • Applied Biosystems 7500 Fast Real‐Time PCR system

Basic Protocol 4: Viral Growth Curves

  Materials
  • WNV New York 1999 seed stock (BEI Resources; http://www.beiresources.org)
  • Cells (see Table 15.3.2) and appropriate medium
  • DMEM + 2% FBS (optional)
  • Phosphate‐buffered saline (PBS) (optional; appendix 2A)
  • Additional reagents and equipment for quantification of WNV by plaque assay ( protocol 2) or focus‐forming assay ( protocol 5)

Alternate Protocol 1: Quantification of WNV by Focus‐Forming Assay

  Materials
  • Vero cells (see protocol 6)
  • Methylcellulose overlay (see recipe)
  • Virus to be titered (from protocol 1 or protocol 4)
  • DMEM + 2% FBS
  • 20% paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15713‐S), diluted to 1% in PBS
  • Phosphate‐buffered saline (PBS), without calcium and magnesium ( appendix 2A)
  • PermWash (see recipe)
  • Primary antibody, e.g., mouse anti‐WNV E16 MAb (Oliphant et al., )
  • Secondary antibody, goat anti‐mouse horseradish‐peroxidase conjugated (Sigma, cat. no. 8924)
  • TrueBlue peroxidase substrate (KPL, cat. no. 50‐78‐02)
  • 96‐well flat‐bottom plates
  • 96‐well U‐bottom plates
  • Multichannel pipettor
  • BioTek 405 SelectTS plate washer (optional)
  • Automated spot counter (e.g., CTL‐BioSpot Analyzer) (optional)

Support Protocol 1: Culturing BHK21 and Vero cells for WNV Propagation and Titering

  Materials
  • BHK21 or Vero cells (available from ATCC or the authors)
  • Phosphate buffered saline (PBS), without calcium or magnesium ( appendix 2A)
  • 0.05% trypsin‐EDTA (Life Technologies, cat. no. 25300054)
  • BHK21 or Vero cell medium (see reciperecipes)
  • Tissue culture–treated flasks
  • 37°C humidified chamber with 5% CO 2

Support Protocol 2: Propagation of C6/36 Insect Cells

  Materials
  • C6/36 cells (ATCC CRL‐1660)
  • C6/36 medium (see recipe)
  • Cell scraper
  • Tissue culture–treated flasks
  • 28°C humidified chamber with 5% CO 2
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Figures

Videos

Literature Cited

Literature Cited
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