Methods Used to Study Respiratory Virus Infection

Emilio Flaño1, Nancy A. Jewell1, Russell K. Durbin1, Joan E. Durbin1

1 Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.3
DOI:  10.1002/0471143030.cb2603s43
Online Posting Date:  June, 2009
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Abstract

This unit describes protocols for infecting the mouse respiratory tract, and assaying virus replication and host response in the lung. Respiratory infections are the leading cause of acute illness worldwide, affecting mostly infants and children in developing countries. The purpose of this unit is to provide a basic strategy and protocols to study the pathogenesis and immunology of respiratory virus infection using the mouse as an animal model. The procedures include: (1) basic techniques for mouse infection, tissue sampling, and preservation, (2) determination of viral titers, isolation and analysis of lymphocytes and dendritic cells using flow‐cytometry, and (3) lung histology, immunohistochemistry, and in situ hybridization. Curr. Protoc. Cell Biol. 43:26.3.1‐26.3.28. © 2009 by John Wiley & Sons, Inc.

Keywords: respiratory viruses; pathology; immunohistochemistry; immunology; respiratory syncytial virus; influenza; murine γ‐herpesvirus 68; lung; lymphoid organs; T lymphocytes; dendritic cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Intranasal Infection of MICE
  • Alternate Protocol 1: Infection of the Uppermost Airway
  • Basic Protocol 2: Determining Virus Titer: Plaque Assay
  • Alternate Protocol 2: Determining Virus Titer: Fluorescent Focus Assay
  • Support Protocol 1: Chemical Sterilization
  • Basic Protocol 3: Leukocyte Isolation
  • Support Protocol 2: Percoll Gradient for Lung Tissue
  • Support Protocol 3: Collagenase Digestion
  • Basic Protocol 4: Flow Cytometry Staining and Analysis of Antigen‐Specific CD8 T Cell Lymphocytes in Lung
  • Alternate Protocol 3: Flow Cytometry Staining and Analysis of Dendritic Cells in Lung
  • Basic Protocol 5: Histology
  • Basic Protocol 6: BAL Cytology
  • Basic Protocol 7: Immunohistochemistry
  • Support Protocol 4: Maximizing Signal: Antigen Retreival
  • Alternate Protocol 4: IHC Using Frozen Sections
  • Basic Protocol 8: In Situ Hybridization
  • Support Protocol 5: Preparation of Avertin Solution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Intranasal Infection of MICE

  Materials
  • Mice
  • Avertin anesthesia (see protocol 17)
  • Viral inoculum diluted in PBS (see recipe) or HBSS (Cellgro) to a volume of 30 to 100 µl/dose
  • 1‐ml syringes and 25‐ to 27‐G needles (BD)
  • Positive displacement pipets and tips (Rainin)

Alternate Protocol 1: Infection of the Uppermost Airway

  Materials
  • Dry ice
  • Mice
  • 70% ethanol
  • Decarbonated Dulbecco's modified Eagle medium (dcDMEM; see recipe)
  • Susceptible cell line
  • 10 mM HEPES supplemented with 5% fetal calf serum
  • Earle's balanced salt solution (Invitrogen) supplemented with 10 mM, pH 7.4 (HEBSA)
  • Overlay medium (see recipe)
  • Fixative/stain (see recipe)
  • 2‐liter beaker (Nalgene)
  • Styrofoam insert (3‐ to 5‐cm thick, equal to the beaker in circumference)
  • Parafilm
  • Dissecting scissors, forceps
  • 5‐ml sterile, plastic snap‐cap tubes, preweighed
  • Tissue homogenizer (e.g., PowerGen model 125, Fisher Scientific)
  • Scale
  • 24‐well plates
  • 37°C, 5% CO 2 incubator
  • 96‐well multiwell plate
  • Dissecting microscope

Basic Protocol 2: Determining Virus Titer: Plaque Assay

  • Virus‐infected cells (see protocol 3, step 10)
  • PBSTA (see recipe)
  • Primary antibody against viral antigen (e.g., rabbit anti‐influenza)
  • Fluorophore‐tagged secondary antibody (e.g., FITC‐goat anti‐rabbit IgG)
  • PBS (see recipe)
  • Inverted fluorescent microscope

Alternate Protocol 2: Determining Virus Titer: Fluorescent Focus Assay

  Materials
  • 0.1% (w/v) SDS/0.006 M sodium hypochlorite (commercial chlorine bleach diluted 1:100)
  • 0.1% (w/v) SDS/0.001% (w/v) Coomassie blue
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; see recipe), sterile
  • Homogenizer
  • Sterile 50‐ml tissue culture tubes

Support Protocol 1: Chemical Sterilization

  Materials
  • Mice
  • Avertin solution (see protocol 17)
  • 70% ethanol
  • HBSS/heparin: one vial heparin (Sigma) to 50 ml HBSS
  • HBSS without MgCl 2, MgSO 4, or CaCl 2 (Cellgro)
  • 5 mg/ml collagenase A in MEM (see recipe)
  • Gey's solution (see recipe)
  • Trypan blue (MP Biomedicals)
  • 80% Percoll (see recipe)
  • Assorted 1‐ml syringes and needles (BD)
  • 15‐ and 50‐ml centrifuge tubes (BD)
  • Forceps, scissors
  • Terumo Surflo i.v. catheter 186 × 1¼–in.
  • 70‐µm cell strainers (BD Falcon)
  • Tissue culture dishes (100‐mm; Falcon)
  • 37°C water bath
  • Centrifuge (e.g., Sorvall Legend RT)
  • Hemacytometer (Hausser Scientific)
  • Microscope (e.g., Zeiss Axiostar plus)

Basic Protocol 3: Leukocyte Isolation

  Materials
  • Percoll (GE Health Care) solutions (see recipe)
  • HBSS without MgCl 2, MgSO 4, or CaCl 2 (Cellgro)
  • Vortexer
  • Centrifuge
  • Pasteur pipets
  • 50‐ml conical tubes

Support Protocol 2: Percoll Gradient for Lung Tissue

  Materials
  • Organ (spleen or lung)
  • Collagenase A solution (see recipe)
  • HBSS
  • HBSS/2 mM EDTA solution (see recipe)
  • Percoll
  • Tissue culture dishes (60‐mm)
  • 1‐ and 3‐ml syringes
  • Razor blades
  • 37°C tissue culture incubator
  • 50‐ml tubes
  • Cell strainer (70‐µm)
  • Cell scraper
  • Centrifuge

Support Protocol 3: Collagenase Digestion

  Materials
  • Single‐cell suspension from lung, spleen, or lymph nodes (see protocol 6 or protocol 8)
  • Antibodies: Fc‐Block (anti‐mouse CD16/CD32 Fc II/III), anti‐CD8α (FITC, Alexa 700, APC), anti‐KLRG1 FITC (eBiosciences or BD Biosciences)
  • Staining wash buffer (SWB; see recipe)
  • Tetrameric reagent: γHV68 ORF6 487‐495 /Kb APC (NIH Tetramer Core Laboratory)
  • 10% (w/v) paraformaldehyde (see recipe)
  • 96‐well round‐bottom plates
  • Centrifuge with plate rotor
  • 1.1‐ml microtubes (National Scientific Supply Co.)
  • Flow cytometer (e.g., FlowJo, TreeStar)

Basic Protocol 4: Flow Cytometry Staining and Analysis of Antigen‐Specific CD8 T Cell Lymphocytes in Lung

  Materials
  • Mice
  • 10% (v/v) buffered formalin (Fisher Scientific)
  • Tissue cassettes (Fisher Scientific)
  • No. 2 pencil
  • Histology laboratory
  • Additional reagents and equipment for removing lungs (see protocol 3)

Alternate Protocol 3: Flow Cytometry Staining and Analysis of Dendritic Cells in Lung

  Materials
  • BAL fluid (see protocol 6)
  • Acetone (Fisher Scientific), cold
  • TBS (see recipe)
  • Hemacytometer
  • Microscope
  • Cytoslides (Thermo Scientific)
  • Disposable cytofunnels (Thermo Scientific)
  • Cytocentrifuge (Thermo Scientific)

Basic Protocol 5: Histology

  Materials
  • 4‐µm tissue sections on Plus slides (Surgipath)
  • Xylenes
  • 95% and 100% ethanol
  • 3% (v/v) H 2O 2 (McKesson)
  • Superblock (Scytek)
  • TBS (see recipe)
  • Primary antibody (determined by investigator)
  • Primary antibody dilution buffer (Biomedia/Fisher Scientific) or TBS (see recipe) with 1% (w/v) BSA (fraction V, Fisher Scientific)
  • Biotinylated secondary antibody (determined by investigator)
  • Streptavidin‐enzyme and substrate kits (Scytek or Dako)
  • Mayer's hematoxylin (Dako)
  • 0.25% (v/v) ammonium hydroxide
  • Crystal/Mount (Biomedia)
  • Permaslip (Alban Scientific)
  • 65°C oven
  • Coplin jars (Fisher Scientific)
  • Pap pen (Biocare Medical)
  • Humidified chamber
  • Coverslips

Basic Protocol 6: BAL Cytology

  Materials
  • 4‐µm section slides
  • 0.5 M Tris⋅Cl, pH 10 (see recipe)
  • Pressure cooker (any)
  • Additional reagents and equipment for deparaffinizing slides (see protocol 13)

Basic Protocol 7: Immunohistochemistry

  Materials
  • Mice
  • Tissue Tek Cryo‐OCT embedding compound (Fisher Scientific)
  • Acetone (Fisher Scientific), cold
  • Desiccant
  • TBS (see recipe)
  • Terumo Surflo i.v. catheter 186 × 1¼‐in.
  • Cryostat
  • Cryostat chuck (obtained from cryostat manufacturer)
  • Plus slides (Surgipath)
  • Coplin jars (Fisher Scientific)

Support Protocol 4: Maximizing Signal: Antigen Retreival

  Materials
  • 4‐µm section slides
  • Diethylpyrocarbonate (DEPC)‐treated water
  • PBS (see recipe)
  • 100 mM glycine (Fisher Scientific)
  • Triton X‐100 (Sigma)
  • 5 µg/ml proteinase K (Sigma) in PBS
  • 4% paraformaldehyde (Electron Microscopy Sciences; see recipe), 4°C
  • 100 mM triethanolamine, pH 8
  • Acetic anhydride (Sigma)
  • Prehybridization buffer (see recipe)
  • Digoxigenin (DIG)‐labeled riboprobe
  • Hybridization buffer (see recipe)
  • 20× SSC (see recipe)
  • ISH buffers 1, 2, and 3 (see reciperecipes)
  • Goat serum (Invitrogen)
  • Anti‐DIG alkaline phosphatase Fab fragment (Roche)
  • NBT/BCIP tablets (Roche)
  • Fast green FCF (see recipe)
  • Crystal/Mount (Biomedia)
  • Pap pen (Biocare Medical)
  • 37°C slide warmer
  • Coplin jars (Fisher Scientific)
  • Humidified chamber (e.g., Tupperware container)
  • 37°, 42°, and 65°C incubator/hybridization oven
  • 80°C heating block
  • 37°C water bath
  • Shaking/orbital rocker at 37°C
  • 15‐ml conical tubes
  • Coverslips
  • Additional reagents and equipment for deparaffinizing slides (see protocol 13)
NOTE: All buffers and reagents should be prepared in DEPC‐treated water.

Alternate Protocol 4: IHC Using Frozen Sections

  Materials
  • Tert‐amyl‐alcohol (Fisher)
  • 2,2,2‐Tribromoethanol (Sigma)
  • 2‐liter glass Erlenmeyer flask
  • Magnetic stirrer
  • Hot plate with stirrer
  • Aluminum foil
  • Bottle‐top filter (0.22‐µm; Millipore)
  • 50‐ml dark glass bottles
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Figures

Videos

Literature Cited

Literature Cited
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   Belz, G.T., Smith, C.M., Kleinert, L., Reading, P., Brooks, A., Shortman, K., Carbone, F.R., and Heath, W.R. 2004. Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I–restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. U.S.A. 101:8670‐8675.
   Cerwenka, A., Morgan, T.M., and Dutton, R.W. 1999. Naive, effector, and memory CD8 T cells in protection against pulmonary influenza virus infection: Homing properties rather than initial frequencies are crucial. J. Immunol. 163:5535‐5543.
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