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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 H₂ O (use for all steps requiring H₂ O)

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% CO₂

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% CO₂

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Figures

Figure 15.D0.1 Crystal violet–stained plaque assay plate. BHK21 cells were infected with 10‐fold dilutions of WNV, overlaid, and stained with crystal violet. Plaque counts were as follows: 10⁻² to 10⁻⁴ , too numerous to count (TNTC); 10⁻⁵ , 118 plaques; 10⁻⁶ , 21 plaques; 10⁻⁷ , 2 plaques. The final viral titer was calculated to be 1.4 × 10⁸ PFU/ml.

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Figure 15.D0.2 Focus‐forming assay. Vero cells were infected with 10‐fold dilutions of WNV. Viral antigen was detected with an anti‐WNV MAb, followed by immunoperoxidase staining (purple). The highest‐concentration samples are in row A, with each row representing a 10‐fold dilution. Six samples were titered in duplicate (in adjacent columns). Viral titers were determined from wells with discrete foci (rows D to F).

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Videos

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited