The Mouse as Model System to Study Host‐Pathogen Interactions in Influenza A Infections

Esther Wilk1, Klaus Schughart1

1 Department of Infection Genetics, Helmholtz Centre for Infection Research and University of Veterinary Medicine Hannover, Braunschweig, Germany
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110173
Online Posting Date:  June, 2012
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The mouse is one of the most important mammalian model systems for studying host‐pathogen‐interactions during influenza A virus infections and for assessing the virulence of newly emerging influenza viruses. Here, we provide the basic protocols for infecting mice with influenza virus and studying the main pathological changes associated with disease. Critical parameters, e.g., virus variants and subtypes or mouse strains, are discussed. Curr. Protoc. Mouse Biol. 2:177‐205 © 2012 by John Wiley & Sons, Inc.

Keywords: infection of mice; influenza A virus; FFU assay; influenza pathology

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Table of Contents

  • Introduction
  • Basic Protocol 1: Virus Propagation in Embryonated Hen's Eggs
  • Basic Protocol 2: Hemagglutination Assay
  • Basic Protocol 3: Titration of Infectious Virus by Focus‐Forming Unit Assay
  • Basic Protocol 4: Intranasal Infection with Influenza A Virus
  • Basic Protocol 5: Weight Loss and Survival
  • Basic Protocol 6: Necropsy and Preparation of Tissue
  • Basic Protocol 7: Bronchoalveolar Lavage (BAL)
  • Basic Protocol 8: Histopathological Analysis of the Infected Lung
  • Basic Protocol 9: Immunohistological Staining of Influenza A Virions
  • Basic Protocol 10: Preparation of RNA from Infected Lungs
  • Support Protocol 1: Virus Propagation in MDCK II Cells
  • Support Protocol 2: Preparation of Red Blood Cell (RBC) Suspension from Chicken Blood
  • Support Protocol 3: Preparation of Lungs for Determination of Viral Load
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Virus Propagation in Embryonated Hen's Eggs

  • SPF (specific‐pathogen‐free) hen's eggs (e.g., from Charles River Laboratories)
  • Influenza virus stock (if titer is known, between 106 and 108 FFU are used for infecting eggs, if titer is unknown, we start with a 1:1000 dilution)
  • Phosphate‐buffered saline (no Mg2+ or Ca2+; Invitrogen)
  • Betaisodona solution/povidone iodide (Mundipharma,
  • Egg incubator (Bruja,
  • Light source/candling box (Orban)
  • Egg punch (autoclavable)
  • 1‐ml syringe
  • Blunt cannula (0.8 × 22 mm, 21‐G × 7/8; Braun)
  • Glue, adhesive tape, or paraffin to seal the hole in the egg (e.g., Ponal wood glue from Henkel;
  • Knife, spoon, and forceps (autoclavable)
  • 15‐ml tubes
  • Pasteur pipets
  • 100‐ to 200‐ml bottle, sterile
  • Additional reagents and equipment for hemagglutination assay ( protocol 2)

Basic Protocol 2: Hemagglutination Assay

  • Allantoic fluid from infected eggs (see protocol 1, step 11) or virus‐containing supernatant from virus propagation on MDCK cells (see protocol 11, step 11)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • 0.9% NaCl (Merck)
  • Chicken red blood cells (RBC) in Alsever's solution (prepare fresh, keep at 4°C; protocol 12)
  • 96‐well V‐bottom microtiter plate

Basic Protocol 3: Titration of Infectious Virus by Focus‐Forming Unit Assay

  • MDCK II cells (Madin Darby Canine Kidney), (ATCC)
  • MDCK II cell culture medium (see recipe)
  • Virus (e.g., harvested from infected embryonated eggs, protocol 1, or lung homogenates, protocol 13)
  • Infection medium (see recipe)
  • Overlay solution (see recipe)
  • Fixative: 4% (v/v) formalin in PBS
  • Quencher solution (see recipe)
  • Blocking buffer (see recipe)
  • Washing buffer: 0.5% (v/v) Tween 20 in phosphate‐buffered saline (PBS; no Ca2+ or Mg2+; Invitrogen)
  • Primary antibody (polyclonal, against influenza A virions, H1N1; Virostat, http://www.virostat‐
  • Secondary antibody (anti‐goat‐IgG‐HRP; KPL,
  • Substrate: TrueBlue peroxidase substrate (KPL,
  • 96‐well plates, flat bottom
  • Infrared heat lamp

Basic Protocol 4: Intranasal Infection with Influenza A Virus

  • Ketamine (100 mg/ml, e.g., Bela‐Pharm GmbH & Co., http://www.bela‐
  • Xylazine (20 mg/ml; cp‐pharma, http://www.cp‐
  • 0.9% (w/v) NaCl (Merck)
  • Influenza A virus (e.g., PR8; see Strategic Planning, Different variants of standard laboratory virus strains) stock solution in PBS
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • 8‐ to 12‐week‐old mice (see Strategic Planning, Choice of mouse strain and age of mice)
  • Eye ointment (Bepanthen, Bayer)
  • 1‐ml syringe and 26‐G, 0.45 × 12 mm needle

Basic Protocol 5: Weight Loss and Survival

  • Animal balance (with box/container/cage to place the mouse in)
  • Software for statistical calculations (e.g., GraphPad Prism 5;
  • Additional reagents and equipment for infection of mice ( protocol 4) and for necroscopy and tissue preparation ( protocol 6)

Basic Protocol 6: Necropsy and Preparation of Tissue

  • Infected mouse (see protocol 4)
  • 70% ethanol
  • Phosphate‐buffered saline (Invitrogen)
  • Optional: Lympholyte M (Cedarlane Laboratories)
  • Digestion medium (see recipe)
  • 2× PBS: dissolve two PBS tablets (Invitrogen) in 500 ml distilled H 2O
  • Surgical instruments including scissors and forceps
  • Winged infusion set (Multifly, Sarstedt)
  • 100‐µm cell strainer (Becton Dickinson)
  • Refrigerated centrifuge
  • 50‐ml tube to accommodate cell strainer
  • 30‐µm filter (CellTrics; Partec,; optional)

Basic Protocol 7: Bronchoalveolar Lavage (BAL)

  • Infected mouse ( protocol 4)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Surgical instruments including scissors and forceps
  • Intravenous indwelling cannula (18‐G, Braun)
  • 1‐ml syringe

Basic Protocol 8: Histopathological Analysis of the Infected Lung

  • Xylol (J.T. Baker)
  • 100%, 90%, and 70% ethanol
  • 1× TBS (TBS tablets, Merck)
  • Paraffin sections of influenza‐infected tissue on slides ( protocol 8)
  • Citrate buffer, pH 6.0 (see recipe)
  • 3% H 2O 2
  • Primary antibody (polyclonal, against influenza A virions, H1N1; Virostat, http://www.virostat‐
  • Antibody diluent (Zytomed Systems)
  • Secondary antibody (biotin‐labeled rabbit α‐goat IgG H+L, KPL,
  • Streptavidin‐HRP (Zytomed Systems)
  • Diaminobenzidine (DAB) substrate buffer (Zytomed Systems)
  • DAB chromogen (Zytomed Systems)
  • Hematoxylin (Merck)
  • Mounting solution (Shandon Consul‐Mount Histology Formulation, Thermo Scientific)
  • Staining jars
  • Coverslips (Menzel)
NOTE: All chemicals must be at room temperature before using them.

Basic Protocol 9: Immunohistological Staining of Influenza A Virions

  • RNAlater RNA stabilization reagent (Qiagen)
  • Infected mouse ( protocol 4)
  • RNase AWAY (Invitrogen)
  • 70% ethanol
  • DEPC‐treated H 2O (see recipe)
  • RNeasy Midi Kit (Qiagen) or equivalent
  • Dissecting instruments
  • Homogenizer (KINEMATICA POLYTRON PT 2100)
  • Centrifuge with a performance of at least 3000 × g
  • NanoDrop spectrophotometer (Thermo Scientific)
  • Agilent 2100 Bioanalyzer (Agilent Technologies)
  • Agilent RNA 6000 Pico/Nano (Agilent Technologies)

Basic Protocol 10: Preparation of RNA from Infected Lungs

  • MDCK II cells (ATCC) growing in culture
  • MDCK II cell culture medium (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • PBS containing 0.2% BSA (Sigma)
  • Virus (e.g., human pandemic 2009 H1N1 isolate)
  • Propagation medium (see recipe)
  • 5 mg/ml TPCK (1‐tosylamide‐2‐phenylethyl chloromethyl ketone)‐Trypsin (Sigma)
  • Liquid N 2 (optional)
  • 75‐cm2 flasks (Biochrom;
  • Additional reagents and equipment for cell culture (Phelan, ) and hemagglutination test ( protocol 2)

Support Protocol 1: Virus Propagation in MDCK II Cells

  • Chicken erythrocytes in Alsever's solution (Fiebig Nährstofftechnik, http://www.fiebig‐
  • 0.9% NaCl
  • Centrifuge
  • Neubauer hemacytometer

Support Protocol 2: Preparation of Red Blood Cell (RBC) Suspension from Chicken Blood

  • Infected mouse ( protocol 4)
  • 70% ethanol
  • PBS/0.1%BSA
  • 14‐ml tubes (round bottom)
  • Homogenizer (KINEMATICA POLYTRON PT 2100)
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