Mammalian Reoviruses: Propagation, Quantification, and Storage

Alicia Berard1, Kevin M. Coombs1

1 University of Manitoba and Manitoba Centre for Proteomics and Systems Biology, Winnipeg, Manitoba, Canada
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.1
DOI:  10.1002/9780471729259.mc15c01s14
Online Posting Date:  August, 2009
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Abstract

Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This unit describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described. Curr. Protoc. Microbiol. 14:15C.1.1‐15C.1.18. © 2009 by John Wiley & Sons, Inc.

Keywords: RNA virus; double‐stranded RNA; non‐enveloped virus; mouse cell culture; cesium chloride ultracentrifugation; plaque assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Mammalian Reoviruses in Cell Culture from Virus Stocks
  • Alternate Protocol 1: Large‐Scale Propagation (and Purification) of Mammalian Reoviruses in Cell Culture from Virus Stocks
  • Basic Protocol 2: Quantification of Mammalian Reoviruses by Plaque Assay with Neutral Red Staining
  • Alternate Protocol 2: Quantification of Mammalian Reoviruses by Plaque Assay with Crystal Violet Staining
  • Basic Protocol 3: Storage of Mammalian Reoviruses
  • Support Protocol 1: Growth and Maintenance of Mouse L929 Cells
  • Support Protocol 2: Plating L929 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Propagation of Mammalian Reoviruses in Cell Culture from Virus Stocks

  Materials
  • Neutral red–stained plate containing virus plaques (see protocol 3)
  • Gel saline (see recipe)
  • 25‐cm2 tissue culture flask with pre‐attached L929 cell monolayer (see protocol 6)
  • Complete MEM medium (see recipe)
  • Penicillin‐streptomycin (see recipe)
  • Amphotericin B (see recipe)
  • 75‐cm2 tissue culture flask(s) with pre‐attached L929 cell monolayer (see protocol 6)
  • Cotton‐plugged Pasteur pipets, sterile
  • 1‐dram (dm) or 2‐dm glass vials, sterile
  • Additional reagents and equipment for obtaining neutral red–stained plates containing virus plaques ( protocol 3)
NOTE: All solutions and materials that come into contact with cells must be sterile, with proper aseptic techniques used.NOTE: All cell culture incubations utilize a 37°C incubator with 5% CO 2 unless otherwise stated.

Alternate Protocol 1: Large‐Scale Propagation (and Purification) of Mammalian Reoviruses in Cell Culture from Virus Stocks

  Materials
  • 6.5 × 108 suspension‐grown L929 cells (see protocol 6)
  • Complete MEM media (see recipe)
  • Penicillin‐streptomycin stock solution (see recipe)
  • Amphotericin B (1000×; see recipe)
  • Mammalian reovirus P 2 stock with titer >108 PFU/ml (see protocol 1)
  • Bleach
  • HO buffer (see recipe)
  • 10% (w/v) sodium desoxycholate (DOC)
  • Vertrel‐XF (Dupont Chemicals)
  • 1.2 g/ml and 1.44 g/ml cesium chloride gradient(s) (see reciperecipes)
  • Dialysis buffer (see recipe)
  • Glycerol (optional)
  • 250‐ml conical centrifuge bottles
  • Refrigerated low‐speed (i.e., up to 2000 rpm, or ∼850 × g) centrifuge with rotor for 250‐ml conical centrifuge bottles
  • Glass roller bottle with sterile magnetic stir bar
  • Pipets and pipet tips
  • 33°C spinning water bath (spinning water bath is set up by placing a large flat‐bottom water‐proof acrylic container on top of a multi‐magnetic stirrer; the container should have sufficient room to hold a temperature‐settable immersion heater and one or more glass roller bottles)
  • 30‐ml COREX centrifuge tubes
  • Ice bucket and ice
  • Sonicator with small probe (for ultrasonic disruption of cells)
  • Vortex
  • Parafilm
  • Refrigerated Super‐speed (i.e., up to 15,000 rpm, or ∼21,000 × g) centrifuge with rotor and adaptors for 30 ml COREX tubes
  • SW‐28 “ultra‐clear”‐type ultracentrifuge tubes
  • Refrigerated ultracentrifuge (i.e., up to 25,000 rpm, or ≥87,000 × g) with swinging‐bucket rotor for SW‐28 ultracentrifuge tubes
  • Dialysis tubing (e.g., Spectra/por 50,000 MWCO) and clips
  • UV spectrophotometer
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 4A)
NOTE: These protocols are for each 1‐liter infection. They can be scaled up or down. For example, we have carried out 6‐liter infections in 12‐liter Florence flasks.

Basic Protocol 2: Quantification of Mammalian Reoviruses by Plaque Assay with Neutral Red Staining

  Materials
  • Pre‐attached L929 cell monolayers in 12‐ or 6‐well plates or 60‐mm dishes (see protocol 7)
  • Gel saline (see recipe)
  • Mammalian reovirus stock/sample to be titrated (the virus may be initially obtained from the ATCC or clinical samples)
  • 2% (w/v) agar (Difco Bacto)
  • Complete 2× 199 medium (see recipe)
  • Amphotericin B (1000×; see recipe)
  • 2% (w/v) neutral red solution (in dH 2O)
  • 2× PBS ( appendix 2A)
  • Penicillin‐streptomycin (100×; see recipe), optional
  • Dilution tubes
  • Micropipettors
  • 100‐ to 1000‐µl pipet tips
  • 20‐ to 200‐µl pipet tips
  • 10‐ml pipets
  • Vortex
  • Autoclavable waste container
  • Microwave
  • 62°C water bath

Alternate Protocol 2: Quantification of Mammalian Reoviruses by Plaque Assay with Crystal Violet Staining

  • 2% (v/v) formaldehyde solution (37% formaldehyde in ddH 2O)
  • Distilled water
  • 2% crystal violet stain (in methanol)
  • Small metal scoop
  • Paper towels

Basic Protocol 3: Storage of Mammalian Reoviruses

  Materials
  • Mouse L929 cells grown in either 25‐, 75‐, or 150‐cm2 tissue culture flask, or in a suspension culture flask (see protocol 1)
  • PBS/EDTA (see recipe)
  • Trypsin (see recipe)
  • Complete MEM medium (see recipe)
  • 5‐ml and 10‐ml pipets
  • 500‐ to 1000‐ml glass flat‐bottom “Florence” flask (for suspension culture)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 4A)
NOTE: All solutions and materials coming into contact with cells must be sterile, with proper aseptic techniques used.NOTE: All cell culture incubations utilize a 37°C incubator at 5% CO 2, except the suspension cultures, which are maintained in a 37°C non‐CO 2 incubator.

Support Protocol 1: Growth and Maintenance of Mouse L929 Cells

  Materials
  • L929 cells in suspension (see protocol 6)
  • Complete MEM medium (see recipe)
  • 5‐ and 10‐ml pipets
  • Appropriate‐sized vessels
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Figures

Videos

Literature Cited

Literature Cited
   Breun, L.A., Broering, T.J., McCutcheon, A.M., Harrison, S.J., Luongo, C.L., and Nibert, M.L. 2001. Mammalian reovirus L2 gene and Lambda2 core spike protein sequences and whole‐genome comparisons of reoviruses type 1 Lang, type 2 Jones, and type 3 Dearing. Virology 287:333‐348.
   Coombs, K.M. 1998a. Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles. Virology 243:218‐228.
   Coombs, K.M. 1998b. Temperature‐sensitive mutants of reovirus. Curr. Topics Microbiol. Immunol. 233:69‐107.
   Dryden, K.A., Coombs, K.M., and Yeager, M. 2008. The structure of orthoreoviruses. In Segmented Double‐stranded RNA Viruses: Structure and Molecular Biology (J.T. Patton, ed.) pp. 3‐25. Horizon Press, Norwich, U.K.
   Hermann, L., Embree, J., Hazelton, P., Wells, B., and Coombs, K. 2004. Reovirus type 2 isolated from cerebrospinal fluid. Ped. Infect. Dis. J. 23:373‐375.
   Johansson, P.J., Sveger, T., Ahlfors, K., Ekstrand, J., and Svensson, L. 1996. Reovirus type 1 associated with meningitis. Scand. J. Infect. Dis. 28:117‐120.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Schiff, L.A., Nibert, M.L., and Tyler, K.L. 2007. Orthoreoviruses and their replication. In Fields Virology (D.M. Knipe and P.M. Howley, eds.) pp. 1853‐1915. Lippincott, Williams & Wilkins, Philadelphia.
   Smith, R.E., Zweerink, H.J., and Joklik, W.K. 1969. Polypeptide components of virions, top component and cores of reovirus type 3. Virology 39:791‐810.
   Starnes, M.C. and Joklik, W.K. 1993. Reovirus protein lambda 3 is a poly(C)‐dependent poly(G) polymerase. Virology 193:356‐366.
   Tyler, K.L., Barton, E.S., Ibach, M.L., Robinson, C., Campbell, J.A., O'Donnell, S.M., Valyi‐Nagy, T., Clarke, P., Wetzel, J.D., and Dermody, T.S. 2004. Isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis. J. Infect. Dis. 189:1664‐1675.
   Wiener, J.R., Bartlett, J.A., and Joklik, W.K. 1989. The sequences of reovirus serotype 3 genome segments M1 and M3 encoding the minor protein mu 2 and the major nonstructural protein mu NS, respectively. Virology 169:293‐304.
   Yin, P., Keirstead, N.D., Broering, T.J., Arnold, M.M., Parker, J.S.L., Nibert, M.L., and Coombs, K.M. 2004. Comparisons of the M1 genome segments and encoded µ2 proteins of different reovirus isolates. Virol. J. 1:6.
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