The Mouse Model for Influenza

Yumiko Matsuoka1, Elaine W. Lamirande1, Kanta Subbarao1

1 NIAID, NIH, Laboratory of Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15G.3
DOI:  10.1002/9780471729259.mc15g03s13
Online Posting Date:  May, 2009
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Abstract

A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and protective immune response to influenza infection in humans. Ferrets are exquisitely susceptible to infection with human influenza viruses and are widely believed to be the ideal small animal model for influenza research. Mice have also been used for influenza vaccine research for decades. Although human influenza viruses generally cause disease in mice only if they are first adapted to the species, the ready availability of mice, their relatively low cost, and the variety of genetic backgrounds and targeted defects, and the immunologic reagents available make the mouse an attractive and heavily utilized animal model for studies of influenza. Although they are not discussed in detail in this unit, hamsters, guinea pigs, cotton rats (Sigmodon), and rats (Rattus) have also been used for influenza research. Curr. Protoc. Microbiol. 13:15G.3.1‐15G.3.30. © 2009 by John Wiley & Sons, Inc.

Keywords: influenza; animal models; mice; virus titration

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isoflurane Anesthesia of Mice
  • Alternate Protocol 1: Alternative Application of Isoflurane
  • Basic Protocol 2: Injectable Anesthesia for Mice
  • Basic Protocol 3: Intranasal Administration of Virus
  • Basic Protocol 4: Measuring Virulence of Virus in Mice as Fifty Percent Mouse Lethal Dose (MLD50)
  • Basic Protocol 5: Blood Collection from the Tail by Incision
  • Basic Protocol 6: Blood Collection from the Retro‐Orbital Sinus
  • Basic Protocol 7: Blood Collection by the Mandibular (Facial Vein/Artery) Approach
  • Basic Protocol 8: Blood Collection from the Heart
  • Basic Protocol 9: Euthanasia Using CO2 and Cervical Dislocation
  • Basic Protocol 10: Mouse Tissue Harvest
  • Basic Protocol 11: Tissue Homogenization
  • Basic Protocol 12: Titration of Tissue Homogenates on MDCK Cells
  • Support Protocol 1: Preparation of MDCK Cells
  • Basic Protocol 13: Titration of Tissue Homogenates in Embryonated Eggs
  • Basic Protocol 14: Measuring Infectivity of Virus in Mice as Fifty Percent Mouse Infectious Dose (MID50)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isoflurane Anesthesia of Mice

  Materials
  • Isoflurane
  • BALB/c mice, 6‐ to 8‐week‐old, female
  • Induction chamber
  • Precision vaporizer (Viking Medical; Fig. )

Alternate Protocol 1: Alternative Application of Isoflurane

  Materials
  • BALB/c mice, 6‐ to 8‐week‐old, female
  • 1‐ml syringes
  • 25‐G needles containing appropriate anesthetic (see Table 15.3.2)

Basic Protocol 2: Injectable Anesthesia for Mice

  Materials
  • Anesthetized mice
  • Virus at appropriate dilution to be inoculated
  • Pipettor (appropriate for 50 µl volume)
  • Pipet tips (appropriate for 50 µl volume)

Basic Protocol 3: Intranasal Administration of Virus

  Materials
  • Dilution medium or buffer (e.g., L‐15 medium, PBS, or other appropriate medium)
  • Virus stock
  • Disinfectant
  • Wet ice
  • Lightly anesthetized BALB/c mice, 6‐ to 8‐week‐old, female; at least four mice per dilution (see protocol 1)
  • Dilution tubes (1.5‐ml microtubes)
  • Pipettor
  • Pipet tips
  • Animal scale
  • Container to hold mice on scale
  • Additional reagents and equipment for inoculating the mouse intranasally ( protocol 4) and euthanizing the animal ( protocol 10)

Basic Protocol 4: Measuring Virulence of Virus in Mice as Fifty Percent Mouse Lethal Dose (MLD50)

  Materials
  • Mice
  • 70% alcohol
  • Heat lamp
  • Mouse restraining box
  • Scalpel
  • Blood collection tube
  • Gauze
  • Tissue glue (optional)

Basic Protocol 5: Blood Collection from the Tail by Incision

  Materials
  • Mice
  • Topical anesthetic (e.g. tetracaine)
  • Microhematocrit tube or Pasteur pipet
  • Blood collection tube
  • Gauze

Basic Protocol 6: Blood Collection from the Retro‐Orbital Sinus

  Materials
  • Mice
  • 21‐, 22‐, or 23‐G needle
  • Blood collection tube
  • Gauze

Basic Protocol 7: Blood Collection by the Mandibular (Facial Vein/Artery) Approach

  Materials
  • Deeply anesthetized mouse (see protocol 1 or protocol 32)
  • 1‐ml syringe equipped with a 22‐G needle
  • Blood collection tube
  • Gauze
  • Additional reagents and equipment for anesthetizing mice ( protocol 1 or protocol 3) and euthanizing mice with CO 2 and/or cervical dislocation ( protocol 10)

Basic Protocol 8: Blood Collection from the Heart

  Materials
  • Mice in cages
  • Absorbent bench paper
  • Lid for cage with tube connected to a CO 2 outlet and regulator
  • Pen or rod‐shaped piece of metal

Basic Protocol 9: Euthanasia Using CO2 and Cervical Dislocation

  Materials
  • 70% ethanol
  • Dry ice or wet ice
  • 10% formalin (at least 15 vol of formalin to 1 vol of tissue)
  • Preweighed collection tubes (e.g., 15‐ml conical tubes)
  • Plastic tri‐pour beakers
  • Surgical instruments including:
    • Scissors
    • Forceps
  • Surgical sutures
  • Tube rack
  • 1‐ to 3‐ml syringes equipped with 20‐G needles
  • Artery clamp
  • Additional reagents and equipment for euthanizing the mice ( protocol 10)

Basic Protocol 10: Mouse Tissue Harvest

  Materials
  • Wescodyne solution (Steris)
  • Tissue samples in 15‐ml or 50‐ml conical tubes (see protocol 11)
  • Wet ice
  • L‐15 tissue grinding medium (see recipe)
  • Dry ice
  • Cryotubes (Nunc, cat. no. 375418)
  • Autoclave
  • Hard disposable homogenizer tips in autoclavable bags (OMNI, cat. no. 34750‐AC‐16)
  • 1 liter beakers for Wescodyne solution
  • Scale
  • Pipets (5, 10, 25, 50 ml)
  • Pipettor
  • OMNI tissue homogenizer
  • 50‐ml conical tubes
  • Absorbent pads (blue pads)
  • Tube racks

Basic Protocol 11: Tissue Homogenization

  Materials
  • 96‐well plates of MDCK cells (see protocol 14)
  • 24‐well plates of MDCK cells (see protocol 14)
  • Homogenized tissue samples (see protocol 12)
  • Wet ice
  • Wescodyne solution (Steris)
  • Complete medium with TPCK trypsin (see recipe)
  • Dry ice
  • Multichannel pipettors
  • Pipet tips, sterile
  • Absorbent pads
  • 1 liter beakers for Wescodyne solution
  • Rocker
  • Additional reagents and equipment for preparing MDCK cells ( protocol 14)

Basic Protocol 12: Titration of Tissue Homogenates on MDCK Cells

  Materials
  • MDCK cells
  • MDCK growth medium (see recipe)
  • MEM medium (Invitrogen, cat. no. 12360‐038)
  • Complete medium with TPCK trypsin (see recipe)
  • 96‐ and 24‐well plates
  • Container lined with absorbent pads

Support Protocol 1: Preparation of MDCK Cells

  Materials
  • 10‐ to 11‐day‐old embryonated hen's eggs (e.g., Charles River Laboratory)
  • Dilution medium or buffer (e.g., L‐15 medium, PBS, or other appropriate medium)
  • Virus or samples to be titrated
  • Disinfectant
  • Wet ice
  • 70% (v/v) ethanol
  • Glue (Elmer's)
  • Washed red blood cells (0.5% turkey or chicken red blood cells in PBS, or 1% horse in PBS and 0.5% BSA)
  • Egg candler (Brinsea, Egg Lume Candling lamp, cat. no. USF150)
  • 1.5‐ml microtubes (for preparation of dilutions)
  • Vortex
  • Repeat dispenser (with tips of appropriate size for 0.9 ml) or pipettor (with 5‐ to 10‐ml disposable pipets)
  • Egg punch (we use sterile and disposable lancet capillary blood sampling devices available from diabetic supply stores)
  • 1‐ml syringes
  • 22‐G, 1‐in. needles
  • Incubator for eggs (35° to 37°C)
  • Forceps
  • 50‐ to 200‐µl multichannel pipettor
  • 50‐ to 200‐µl pipet tips
  • 96‐well V‐shaped plates

Basic Protocol 13: Titration of Tissue Homogenates in Embryonated Eggs

  Materials
  • Dilution medium or buffer (e.g., L‐15 medium, PBS, or other appropriate medium)
  • Virus stock
  • Disinfectant
  • Ice
  • BALB/c mice, 6‐ to 8‐week‐old, female; four mice per dilution per day
  • 1.5‐ml microtubes (for preparation of dilutions)
  • Pipettor
  • Pipet tips
NOTE: If you are identifying infected mice by virus detection in the lungs at two time points e.g., days 2 and 3, with 10−1 through 10−7 dilutions of virus stock, you will need 4 mice × 7 dilutions × 2 days = 56 mice
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Figures

Videos

Literature Cited

   Abou‐Donia, H., Jennings, R., and Potter, C.W. 1980. Growth of influenza A viruses in hamsters. Arch. Virol. 65:99‐107.
   Andrewes, C.H., Laidlaw, P.P., and Smith, W. 1934. The susceptibility of mice to the viruses of human and swine influenza. Lancet 2:859‐862.
   Brown, E.G., Liu, H., Chang Kit, L., Baird, S., and Nesrallah, M. 2001. Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes. Proc. Natl. Acad. Sci. U.S.A. 98:6883‐6888.
   Epstein, S.L., Lo, C.Y., Misplon, J.A., Lawson, C.M., Hendrickson, B.A., Max, E.E., and Subbarao, K. 1997. Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell‐depleted, β2‐microglobulin‐deficient, and J chain‐deficient mice. J. Immunol. 158:1222‐1230.
   Grimm, D., Staeheli, P., Hufbauer, M., Koerner, I., Martinez‐Sobrido, L., Solorzano, A., Garcia‐Sastre, A., Haller, O., and Kochs, G. 2007. Replication fitness determines high virulence of influenza A virus in mice carrying functional Mx1 resistance gene. Proc. Natl. Acad. Sci. U.S.A. 104:6806‐6811.
   Heath, A.W., Addison, C., Ali, M., Teale, D., and Potter, C.W. 1983. In vivo and in vitro hamster models in the assessment of virulence of recombinant influenza viruses. Antiviral Res. 3:241‐252.
   Iida, T. and Bang, F.B. 1963. Infection of the upper respiratory tract of mice with influenza virus. Am. J. Hyg. 77:169‐176.
   Lowen, A.C., Mubareka, S., Tumpey, T.M., Garcia‐Sastre, A., and Palese, P. 2006. The guinea pig as a transmission model for human influenza viruses. Proc. Natl. Acad. Sci. U.S.A. 103:9988‐9992.
   Luke, C.J. and Subbarao, K. 2008. The role of animal models in influenza vaccine research. In Influenza Vaccines for the Future, Series: Advances in Infectious Diseases (R. Rappuoli and G. Del Giudice, eds) pp. 161‐202. Birkhäuser Verlag, Basel, Switzerland.
   Niewiesk, S. and Prince, G. 2002. Diversifying animal models: The use of hispid cotton rats (Sigmodon hispidus) in infectious diseases. Lab. Anim. 36:357‐372.
   Ottolini, M.G., Blanco, J.C., Eichelberger, M.C., Porter, D.D., Pletneva, L., Richardson, J.Y., and Prince, G.A. 2005. The cotton rat provides a useful small‐animal model for the study of influenza virus pathogenesis. J. Gen. Virol. 86:2823‐2830.
   Phair, J.P., Kauffman, C.A., Jennings, R., and Potter, C.W. 1979. Influenza virus infection of the guinea pig: Immune response and resistance. Med. Microbiol. Immunol. 165:241‐254.
   Reed, L.J. and Muench, H. 1938. Simple method of estimating fifty percent endpoints. Amer. J. Hygiene 27:493‐497.
   Sidwell, R.W., Huffman, J.H., Gilbert, J., Moscon, B., Pedersen, G., Burger, R., and Warren, R.P. 1992. Utilization of pulse oximetry for the study of the inhibitory effects of antiviral agents on influenza virus in mice. Antimicrob. Agents Chemother. 36:473‐476.
   Spackman, E. and Suarez, D.L. 2008. Type A influenza virus detection and quantitation by real‐time RT‐PCR. Methods Molec. Biol. 436:19‐26.
   Staeheli, P., Grob, R., Meier, E., Sutcliffe, J.G., and Haller, O. 1988. Influenza virus‐susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol. Cell. Biol. 8:4518‐4523.
   Subbarao, E.K., Kawaoka, Y., and Murphy, B.R. 1993. Rescue of an influenza A virus wild‐type PB2 gene and a mutant derivative bearing a site‐specific temperature‐sensitive and attenuating mutation. J. Virol. 67:7223‐7228.
   Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., and Murphy, B. 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome corona virus in the respiratory tract of mice. J. Virol. 78:3572‐3577.
   Takiguchi, K., Sugawara, K., Hongo, S., Nishimura, H., Kitame, F., and Nakamura, K. 1992. Protective effect of serum antibody on respiratory infection of influenza C virus in rats. Arch. Virol. 122:1‐11.
   Teh, C., Jennings, R., and Potter, C.W. 1980. Influenza virus infection of newborn rats: Virulence of recombinant strains prepared from influenza virus strain A/Okuda/57. J. Med. Microbiol. 13:297‐306.
   van der Laan, J.W., Herberts, C., Lambkin‐Williams, R., Boyers, A., Mann, A.J., and Oxford, J. 2008. Animal models in influenza vaccine testing. Expert Rev. Vaccines 7:783‐793.
   Van Riel, D., Munster, V.J., de Wit, E., Rimmelzwaan, G.F., Fouchier, R.A.M., Osterhaus, A.D.M.E., and Kuiken, T. 2006. H5N1 virus attachment to lower respiratory tract. Science 312:399.
   Yetter, R.A., Lehrer, S., Ramphal, R., and Small, P.A.J. 1980. Outcome of influenza infection: Effect of site of initial infection and heterotypic immunity. Infect. Immun. 29:654‐662.
Key References
   Queensbury, K.E. and Carpenter, J.W. (eds.) 2004. Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery, 2nd edition. Saunders, St. Louis, Mo.
  This book discusses the anatomy, husbandry, physiology, and diseases of small mammals including ferrets, rabbits, guinea pigs, and other small rodents.
   Hedrich, H.J. and Bullock, G. (eds.) 2006. The Laboratory Mouse. Elsevier Academic Press, London.
  This book covers mouse genetics, animal husbandry and production, animal procedures, normal histology, and pathophysiology.
   van der Laan et al., 2008. See above.
  This review article discusses various animal species that can be experimentally infected with influenza.
   Luke and Subbarao, 2008. See above.
  This chapter discusses the evaluation of influenza virus vaccines in animal models.
Internet Resources
   http://www.cdc.gov/od/sap
  CDC Select Agent program.
   http://www.avma.org/resources/euthanasia.pdf
  Euthanasia guidelines from American Veterinary Medical Association.
   http://www3.niaid.nih.gov/labs/aboutlabs/cmb/InfectiousDiseasePathogenesisSection/mouseNecropsy/
  Mouse necropsy procedure.
   http://www.searo.who.int/LinkFiles/CDS_CDS‐Guidelines‐Laboratory.pdf
  WHO Guidelines on Laboratory Diagnosis of Avian Influenza (2007).
   http://www.wpro.who.int/NR/rdonlyres/EFD2B9A7‐2265‐4AD0‐BC98‐97937B4FA83C/0/manualonanimalaidiagnosisandsurveillance.pdf
  WHO Manual on Animal Influenza Diagnosis and Surveillance.
   http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
  5th edition of Biosafety in Microbiological and Biomedical Laboratories.
   http://www.cdc.gov/flu/pdf/h2n2bsl3/pdf
  Biosafety guidelines for noncontemporary or nonhuman influenza viruses from the 5th edition of Biosafety in Microbiological and Biomedical Laboratories.
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