Influenza Virus

Robert Cottey1, Cheryl A. Rowe1, Bradley S. Bender1

1 University of Florida College of Medicine, Gainesville, Florida
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.11
DOI:  10.1002/0471142735.im1911s42
Online Posting Date:  May, 2001
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Abstract

This unit contains several methods for infecting mice with influenza virus. It also includes protocols needed to propagate influenza virus in hen eggs, quantitate virus titers (in tissue culture medium and in influenza‐infected mouse serum), and adopt human isolates of influenza for growth in mice. Methods for measuring the 50% mouse lethal dose are also included. Finally, protocols for generating anti‐influenza cytotoxic T lymphocytes (CTL) from splenocyte precursors and harvesting pulmonary CTL following respiratory tract challenge of mice with influenza virus are provided.

     
 
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Table of Contents

  • Basic Protocol 1: Intranasal Infection of Anesthetized Mice with Influenza Virus
  • Alternate Protocol 1: Intranasal Infection of Unanesthetized Mice with Influenza Virus
  • Support Protocol 1: Propagation of Influenza Virus in Hen Eggs
  • Support Protocol 2: Quantitation of Influenza Virus Titer in Egg Allantoic Fluid
  • Support Protocol 3: Long‐Term Culture of MDCK Cells
  • Support Protocol 4: Measurement of Tissue Culture Influenza Virus Infectious Dose (TCID50)
  • Basic Protocol 2: Creation of a Mouse‐Adapted Human Influenza Virus
  • Support Protocol 5: Determination of 50% Mouse Lethal Dose (MLD50) and 50% Mouse Infectious Dose (MID50)
  • Support Protocol 6: Preparation of Mouse Lungs for Measurement of Influenza Virus Titers
  • Support Protocol 7: Collection of Mucosal Fluid from the Mouse Nose and Vagina
  • Support Protocol 8: Determination of the Number of Hemagglutination Units (HAU) in an Influenza Virus Stock
  • Support Protocol 9: Hemagglutination Inhibition (HAI) Assay Using Mouse Serum
  • Support Protocol 10: Serial Dilution of an Influenza Stock Virus
  • Basic Protocol 3: Generation of Anti‐Influenza Cytotoxic T‐Lymphocytes (CTL) from Splenic Precursors
  • Basic Protocol 4: Harvesting Pulmonary Cells Containing Anti‐Influenza CTL Activity
  • Support Protocol 11: Influenza Virus Neutralization Assay
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Intranasal Infection of Anesthetized Mice with Influenza Virus

  Materials
  • 6‐ to 8‐week‐old mouse (e.g. BALB/c)
  • 1× 102 to 1 × 105 TCID 50/ml influenza virus (see protocol 4 and protocol 64), titered
  • Additional reagents and equipment for handling and restraint (unit 1.3), and anesthesia using ketamine/xylazine (unit 1.4)

Alternate Protocol 1: Intranasal Infection of Unanesthetized Mice with Influenza Virus

  Materials
  • 9‐ to 11‐day‐old, specific‐pathogen free (SPF), fertile hen's eggs (SPAFAS or Hy‐Vac)
  • 5000 U/5 mg/ml (each) penicillin/streptomycin solution
  • Stock influenza virus at 1 × 103 TCID 50/ml
  • 70% alcohol
  • Candling box (Stromberg's Chicks and Game Birds)
  • 70% alcohol pads
  • Egg punch (Stromberg's Chicks and Game Birds)
  • 1‐ml syringe with 25‐G, 5/8‐in. needle
  • Adhesive tape
  • Egg flats
  • 34° to 35°C egg incubator, 82% humidity (Stromberg's Chicks and Game Birds)
  • Pointed scissors, sterile
  • Metal spatula, sterile
  • 5 3/4‐in. Pasteur pipets, sterile with rubber bulb
  • 50‐ml conical tubes, sterile
  • IEC floor‐mounted, refrigerated, DPR‐6000 centrifuge and 253 rotor or equivalent
  • 0.45‐µm filter (optional)
  • 1‐ml cryovials
  • Additional reagents and equipment for measuring hemagglutinin units (see protocol 11) and 50% tissue culture dose (TCID 50; see protocol 6)

Support Protocol 1: Propagation of Influenza Virus in Hen Eggs

  Materials
  • DMEM‐5 (Clonetics; also see appendix 2A) containing 50 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B
  • Fluid to be tested (e.g., tissue homogenate, allantoic fluid; see protocol 3)
  • 2.5 × 105 Madin‐Darby canine kidney (MDCK) cells (ATCC #CCL‐34; see Internet Resources)
  • DMEM/0.0002% trypsin containing 50 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B
  • 0.5% chicken red blood cell (CRBC) suspension in sterile PBS (see protocol 11, steps to ), fresh
  • 96‐well, round‐bottom, tissue‐culture‐treated plates with lids, sterile
  • Multichannel pipettor
  • Humidified 35°C, 5% CO 2 incubator
  • Tissue homogenizer (Fisher Scientific)
  • Influenza virus of known titer (e.g., A/Port Chalmers/73, ATCC #VR‐810; A/PR/8/34, ATCC# VR‐95)
  • Additional reagents and equipment for measuring viral titers using the TCID 50 assay (see protocol 6) or the viral neutralization assay (see protocol 13), and counting viable cells by trypan blue dye exclusion ( appendix 3B)

Support Protocol 2: Quantitation of Influenza Virus Titer in Egg Allantoic Fluid

  Materials
  • MDCK cells (ATCC# CCL‐34; see Internet Resources)
  • recipeHank's balanced salt solution (HBSS; appendix 2A) without Ca2+ or Mg2+, with and without 0.25% trypsin and 0.03% EDTA
  • DMEM‐5 and ‐10 containing 50 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B
  • 75‐cm2 tissue culture flask
  • Humidified 37°C, 5% CO 2 incubator
  • 50‐ml conical tube, sterile
  • Additional reagents and equipment for counting viable cells ( appendix 3B).

Support Protocol 3: Long‐Term Culture of MDCK Cells

  • Virus samples

Support Protocol 4: Measurement of Tissue Culture Influenza Virus Infectious Dose (TCID50)

  Materials
  • 6‐ to 8‐week‐old mice, 20 to 25 g (e.g. BALB/c)
  • ∼105 TCID 50 influenza A virus stock, titered (see protocol 4 and protocol 64)
  • recipeHBSS, sterile ( appendix 2A) containing 100 U/ml penicillin and 100 µg/ml streptomycin
  • Sterile recipePBS ( appendix 2A)
  • 60 × 15–mm petri dish, sterile
  • Scissors, sterile
  • Forceps, sterile
  • Glass homogenizer (Fisher Scientific), sterile
  • 10‐ml conical tube
  • 100‐ml glass bottle, sterile
  • 5‐ml sterile cryovials (optional)
  • IEC floor‐mounted, refrigerated, DPR‐6000 centrifuge and 253 rotor or equivalent
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4), euthanasia by cervical dislocation (unit 1.8), and determining MLD 50 and MID 50 (see protocol 8)

Basic Protocol 2: Creation of a Mouse‐Adapted Human Influenza Virus

  Materials
  • Titered influenza virus stock (see protocol 6)
  • Mice (e.g. BALB/c 6‐ to 8‐week‐old)
  • 10 mg/0.4 mg/ml ketamine/xylazine anesthetic solution in sterile recipePBS ( appendix 2A)
  • recipePBS, sterile
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4), blood collection from the tail (unit 1.7), and HAI assay (see protocol 13)

Support Protocol 5: Determination of 50% Mouse Lethal Dose (MLD50) and 50% Mouse Infectious Dose (MID50)

  Materials
  • 8‐week‐old influenza‐infected BALB/c mouse (see protocol 1)
  • Sterile recipePBS ( appendix 2A), cold
  • 70% ethanol
  • 16 × 125–mm polypropylene sterile round‐bottom, screw‐cap tube
  • Motor‐driven pestle: 13 × 210–mm Teflon‐coated steel‐shaft pestle (Fisher) and Tri‐R Stir‐R variable speed electric motor (Tri‐R instruments, model K‐41)
  • Tabletop centrifuge
  • 1‐ml sterile cryovials
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4) and intranasal infection of anesthetized mice (see protocol 1)

Support Protocol 6: Preparation of Mouse Lungs for Measurement of Influenza Virus Titers

  Materials
  • Mouse
  • ketamine/xylazine anesthetic solution (unit 1.4)
  • recipePBS ( appendix 2A), sterile with and without 20 µg/ml aprotinin (Boeringer Mannheim)
  • 0.01 M dithiothreitol solution (DTT, Sigma; vaginal)
  • Styrofoam tube rack (i.e., conical‐tube packaging; nasal)
  • 23‐ and B‐bevel 25‐G needles (nasal)
  • Scissors and scalpel (nasal), sterile
  • Hemostats or monofilament nylon (nasal)
  • 1‐ml tuberculin syringe (nasal)
  • 200‐µl pipettor (vaginal)
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4) and TCID 50 assay (see protocol 6).

Support Protocol 7: Collection of Mucosal Fluid from the Mouse Nose and Vagina

  Materials
  • Chicken blood collected 1:1 in Alsever's solution ( appendix 2A) or other anticoagulant
  • recipePBS ( appendix 2A), sterile
  • PBS/0.5% BSA solution
  • Stock influenza virus in allantoic fluid (see protocol 3)
  • 15‐ml and 50‐ml conical tubes, sterile
  • 96‐well round‐bottom microtiter plate
  • Multichannel pipettor
  • recipePBS, sterile ( appendix 2A)

Support Protocol 8: Determination of the Number of Hemagglutination Units (HAU) in an Influenza Virus Stock

  Materials
  • Sera from influenza infected mice
  • 1% CRBC suspension in PBS (see protocol 11, steps to )
  • recipePBS ( appendix 2A)/0.5% BSA solution
  • Stock viral antigen diluted to contain 4 HAU (see protocol 11)
  • 56°C water bath
  • 96‐well, nonsterile, nontissue culture–treated, round‐bottom microtiter plate
  • Multichannel pipettor
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4) for preparation of serum from mouse blood (unit 2.4)

Support Protocol 9: Hemagglutination Inhibition (HAI) Assay Using Mouse Serum

  Materials
  • Influenza virus stock, titered (see Support Protocols protocol 42 and protocol 64)
  • recipePBS, sterile ( appendix 2A)
  • 15‐ml conical tubes, sterile
NOTE: For this example, a stock virus concentration of 107.31 has been chosen arbitrarily.

Support Protocol 10: Serial Dilution of an Influenza Stock Virus

  Materials
  • Influenza A‐infected BALB/c mice, 6 to 8 weeks old (see protocol 1 or protocol 2)
  • Influenza B‐infected BALB/c mice, 6 to 8 weeks old
  • recipePBS, sterile
  • Iscove's/10% FBS medium containing 50 µg/ml gentamicin, 100U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 50 µM recipe2‐mercaptoethanol ( appendix 2A)
  • ∼109 TCID 50 H3N2 influenza A virus (e.g., B/Lee/40; ATTC# VR‐101) in allantoic fluid (see Support Protocols protocol 31, protocol 42 and protocol 64)
  • Glass tissue homogenizer, sterile (Fischer Scientific)
  • 75‐cm2 tissue culture flask
  • 15‐ and 50‐ml polypropylene conical tubes, sterile
  • IEC floor‐mounted, refrigerated, DPR‐6000 centrifuge and 253 rotor or equivalent
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4), preparing single cell suspensions (unit 3.1), measuring CTL activity (unit 3.11), growing P815 cells (unit 4.7), and counting viable cells ( appendix 3B).

Basic Protocol 3: Generation of Anti‐Influenza Cytotoxic T‐Lymphocytes (CTL) from Splenic Precursors

  Materials
  • Influenza‐infected mice (see protocol 1 or protocol 2)
  • PBS ( appendix 2A), sterile
  • Iscove's/10% FBS with and without 50 U/ml collagenase (Sigma)
  • Red blood cell lysing buffer (unit 3.1)
  • 60‐mm petri dish, sterile
  • Scissors, sterile
  • 37°C, 5% CO 2 incubator
  • Tissue grinder kit, with sterile tissue grinder cup, glass pestle, and 60‐mesh screen (e.g., Sigma CD‐1)
  • 15‐ml conical tube, sterile
  • Additional reagents and equipment for anesthesia using ketamine/xylazine (unit 1.4) and for enriching T cell populations through nylon wool columns (unit 3.2).

Basic Protocol 4: Harvesting Pulmonary Cells Containing Anti‐Influenza CTL Activity

  Materials
  • DMEM/5% FBS containing 50 µg/ml gentamicin, 100U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B with and without 2.5 × 105 MDCK cells (ATCC# CCL‐34; see Internet Resources)
  • Serum from influenza‐infected mice (see protocol 1 or protocol 2)
  • Titered influenza virus containing 50 TCID 50/50 µl (see protocol 6)
  • DMEM/0.0002% trypsin containing 50 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B
  • 0.5% CRBC in PBS (Clonetics; also see protocol 11, steps to )
  • 96‐well, sterile round‐bottom, tissue culture–treated, microwell plate with lid
  • Multichannel pipettor
  • 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for anesthesia using ketamine/xylazine in mice (unit 1.4) for preparation of serum from mouse blood (unit 2.4)
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Literature Cited

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   Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., and Kawaoka, Y. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152‐179.
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Key References
   Doherty, P.C., Topham, D.J., Tripp, R.A., Cardin, R.D., Brooks, J.W., and Stevenson, P.G. 1997. Effector CD4+ and CD8+ T‐cell mechanisms in the control of respiratory virus infections. Immunol. Rev. 159:105‐117.
  Authoritative review on recovery from influenza and other respiratory viruses.
   Gerhard, W., Mozdzanowska, K., Furchner, M., Washko, G., and Maiese, K. 1997. Role of the B‐cell response in recovery of mice from primary influenza virus infection. Immunol. Rev. 159:95‐103.
  Authoritative review on recovery from influenza and other respiratory viruses.
   Mahy, B.W.J. and Kangro, H. O. 1996. Virology Methods Manual. Academic Press. San Diego, CA.
  Excellent overview of general virology procedures.
   Reed and Muench, 1938. See above.
  Gives derivation of formula for calculating 50% endpoints.
   Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., and Kawaoka, Y. 1980. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152‐179.
  Thorough discussion of influenza virology, with an emphasis on viral evolution.
   Yetter, R.A., Lehrer, S., Ramphal, R., and Small, P.A., Jr. 1980. Outcome of influenza infection: effect of site of initial infection and heterotypic immunity. Infect. Immun. 29:654‐662.
  Detailed description of the mouse model of influenza.
Internet Resources
   http://www.cdc.gov/ncidod/diseases/flu/fluvirus.htm
  The Centers for Disease Control and Prevention web site for current vaccines, antivirals and influenza surveillance.
   http://www.who.ch/emc/diseases/flu/index.html
  The World Health Organization web site for global influenza surveillance.
   http://www‐micro.msb.le.ac.uk/335/Orthomyxoviruses.html
  These are two on‐line learning modules designed for practicing physicians, students, and other health care workers.
   http://www.medinfo.ufl.edu/cme/flu/flu.html
  This is a learning module for medical students designed to teach the principles behind the serologic diagnosis of influenza by hemagglutination inhibition assay. It requires Adobe Acrobat Reader.
   www.medinfo.ufl.edu/∼pops/mepopsfinal/Influenza.pdf
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