A Mouse Model of West Nile Virus Infection

Jessica B. Graham1, Jessica L. Swarts1, Jennifer M. Lund2

1 Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 2 Department of Global Health, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.33
Online Posting Date:  December, 2017
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Abstract

The use of a mouse model to study the breadth of symptoms and disease severity seen in human West Nile virus (WNV) infection can provide insight into the kinetics of the immune response and the specific pathways responsible for control of WNV infection and viral clearance. Here, we provide protocols for performing WNV infection of mice, as well as complete immunophenotyping analysis of the cellular immune response to infection in both the periphery and the central nervous system in a mouse model of WNV infection. © 2017 by John Wiley & Sons, Inc.

Keywords: clinical disease; immunophenotyping; West Nile virus

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: West Nile Virus Infection via Footpad Injection
  • Basic Protocol 2: Clinical Score and Weight Loss
  • Basic Protocol 3: Tissue Harvest and Processing
  • Basic Protocol 4: Flow Cytometry Phenotyping for Identification of Immune Subsets, Chemokines, and Cytokines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: West Nile Virus Infection via Footpad Injection

  Materials
  • 100 mg/ml ketamine
  • 20 mg/ml xylazine
  • Phosphate‐buffered saline (PBS; e.g., Invitrogen)
  • West Nile virus (WNV; kindly provided by the Gale laboratory, University of Washington)
  • 8‐ to 10‐week‐old mice (see Strategic Planning)
  • 1/2 cc insulin syringe with 28‐G needle (e.g., BD Biosciences)

Basic Protocol 2: Clinical Score and Weight Loss

  Materials
  • Infected mouse (see protocol 1)
  • Hydrogel (water in clear gel form)
  • Balance and animal weigh boat or container
  • Data sheet for recording weight, clinical score, and any notes
  • Data analysis/statistical software (e.g., GraphPad Prism)

Basic Protocol 3: Tissue Harvest and Processing

  Materials
  • Mouse of interest
  • 70% ethanol
  • PBS, sterile (e.g., Invitrogen)
  • RPMI complete medium (see recipe)
  • Hypertonic Percoll (see recipe)
  • 0.5% FACS buffer (see recipe)
  • Trypan blue
  • Bleach
  • ACK lysis buffer (see recipe)
  • CO 2 chamber, for euthanasia
  • Absorbent disposable pads
  • Forceps
  • Surgical scissors
  • 2‐ml microcentrifuge tubes
  • 15‐ml conical tubes
  • 30‐ml syringe
  • 18‐G, 1.5‐in. needles
  • 6‐well plates with 70‐µm cell strainers
  • Frosted microscope slides
  • Vortex
  • Refrigerated centrifuge
  • 70‐μm filter‐top FACS tubes
  • Hemocytometer
  • Microcentrifuge

Basic Protocol 4: Flow Cytometry Phenotyping for Identification of Immune Subsets, Chemokines, and Cytokines

  Materials
  • Single‐cell suspensions (see protocol 3)
  • Dimethyl sulfoxide (DMSO)
  • NS4b peptide, available from Fred Hutchinson Cancer Research Center Immune Monitoring Core
  • HI‐WNV
  • CD3 antibody (e.g., eBioscience, cat. no. 16‐0031‐85)
  • CD28 antibody (e.g., eBioscience, cat. no. 16‐0281‐85)
  • 1000× Brefeldin A Solution (BFA; e.g., eBioscience, cat. no. 00‐4506‐51)
  • RPMI complete medium (see recipe)
  • Bleach, for waste disposal
  • PBS (e.g., Invitrogen)
  • LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (e.g., Thermo Fisher Scientific, cat. no. L34957)
  • CD16/32 antibody (e.g., eBioscience, cat. no. E0359‐1630)
  • 0.5% FACS buffer (see recipe)
  • Antibodies (see Table 17.2.0)
  • Fixation/Permeabilization kit (e.g., eBioscience, cat. no. 88‐8824‐00) containing:
    • IC Fixation Buffer
    • 10× Permeabilization Buffer
  • 96‐well U‐bottom plate
  • 37°C, 5% CO 2 incubator
  • Aluminum foil
  • Centrifuge with plate adaptor
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Figures

Videos

Literature Cited

Literature Cited
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