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

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Virus Propagation in Embryonated Hen's Eggs

Materials

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, http://www.mundipharma.de)

Egg incubator (Bruja, http://www.bruja.de/)
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; http://www.henkel.com/)
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

Materials

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

Materials

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‐inc.com/)
Secondary antibody (anti‐goat‐IgG‐HRP; KPL, http://www.kpl.com/)
Substrate: TrueBlue peroxidase substrate (KPL, http://www.kpl.com/)

96‐well plates, flat bottom
Infrared heat lamp

Basic Protocol 4: Intranasal Infection with Influenza A Virus

Materials

Ketamine (100 mg/ml, e.g., Bela‐Pharm GmbH & Co., http://www.bela‐pharm.com/)
Xylazine (20 mg/ml; cp‐pharma, http://www.cp‐pharma.de/)
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

Materials

Animal balance (with box/container/cage to place the mouse in)
Software for statistical calculations (e.g., GraphPad Prism 5; http://www.graphpad.com/)

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

Materials

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 H₂ O

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, http://www.partec.com; optional)

Basic Protocol 7: Bronchoalveolar Lavage (BAL)

Materials

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

Materials

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% H₂ O₂
Primary antibody (polyclonal, against influenza A virions, H1N1; Virostat, http://www.virostat‐inc.com/)
Antibody diluent (Zytomed Systems)
Secondary antibody (biotin‐labeled rabbit α‐goat IgG H+L, KPL, http://www.kpl.com/)
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

Materials

RNAlater RNA stabilization reagent (Qiagen)
Infected mouse ( protocol 4 )
RNase AWAY (Invitrogen)
70% ethanol
DEPC‐treated H₂ O (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

Materials

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 N₂ (optional)
75‐cm² flasks (Biochrom; http://www.biochrom.co.uk/)

Additional reagents and equipment for cell culture (Phelan, ) and hemagglutination test ( protocol 2 )

Support Protocol 1: Virus Propagation in MDCK II Cells

Materials

Chicken erythrocytes in Alsever's solution (Fiebig Nährstofftechnik, http://www.fiebig‐naehrstofftechnik.de/)
0.9% NaCl

Centrifuge
Neubauer hemacytometer

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

Materials

Infected mouse ( protocol 4 )
70% ethanol
PBS/0.1%BSA

14‐ml tubes (round bottom)
Homogenizer (KINEMATICA POLYTRON PT 2100)

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Figures

Figure 1. Pipetting scheme for performing the hemagglutination assay.

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Figure 2. Body weight loss over time. Mice from different inbred strains were infected intranasally with 2 × 10³ FFU of PR8 virus. The percentage of the starting weight (set to 100%) is plotted for each day post infection. The figure has been reproduced from Srivastava et al. ().

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Figure 3. Kaplan‐Meier survival curves. DBA/2J and C57BL/6J mice were infected with increasing doses of PR8 virus via the intranasal route, and survival was monitored for the following 14 days. For each day, the cumulative number of mice that died or were euthanized is recorded. The cumulative percentage of surviving animals is plotted against the time post infection. The figure has been reproduced from Srivastava et al. ()

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Figure 4. HE staining of lungs. Healthy (A ) and infected lungs (B ) on day 10 after infection of C57BL/6J mice with 2 × 10³ FFU of PR8 virus are shown. Infected lungs show massive infiltration of immune cells into the lung tissue.

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Figure 5. Immunohistochemical staining for viral proteins in infected lungs. C57BL/6J mice were infected intranasally with 2 × 10³ FFU of PR8M virus. Serial lung sections were stained with anti‐influenza antibody and hematoxylin). The spread of PR8M virus (cells stained brown) at day 1 post infection was mostly limited to bronchiolar regions (B ). In noninfected mice, no staining was detected (A ). The figure has been partially reproduced from Blazejewska et al. ()

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Figure 6. Determination of FFU. Image of tissue culture plate (mirror‐inverted) after infection of cells and antibody staining against influenza A proteins. Staining is visualized with TrueBlue peroxidase substrate. Blue foci indicate infected cells.

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Figure 7. Measurement of viral load in infected lungs by FFU. DBA/2J and C57BL/6J were infected intranasally with 2 × 10³ FFU of a low virulent PR8 variant (PR8M) (A ), a more virulent PR8 variant (PR8F) (B ), or a highly virulent PR8 variant (hv PR8) (C ), and viral load in the lungs was determined at indicated days post infection using focus‐forming unit assay. The figure has been reproduced from Blazejewska et al. ()

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

Abstract

Table of Contents

Materials

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

Figures

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Literature Cited