Myxoma Virus: Propagation, Purification, Quantification, and Storage

Sherin E. Smallwood1, Masmudur M. Rahman1, Dorothy W. Smith1, Grant McFadden1

1 College of Medicine, University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14A.1
DOI:  10.1002/9780471729259.mc14a01s17
Online Posting Date:  May, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Myxoma virus (MYXV) is a member of the Poxviridae family and prototype for the genus Leporipoxvirus. It is pathogenic only for European rabbits, in which it causes the lethal disease myxomatosis, and two North American species, in which it causes a less severe disease. MYXV replicates exclusively in the cytoplasm of the host cell. Although not infectious in humans, its genome encodes proteins that can interfere with or modulate host defense mechanisms; it is able to productively infect a number of human cancer cell lines, but not normal human cells, and has also been shown to increase survival time in mouse models of human glioma. These characteristics suggest that MYXV could be a viable therapeutic agent, e.g., in anti‐inflammatory or anti‐immune therapy, or as an oncolytic agent. MYXV is also an excellent model for poxvirus biology, pathogenesis, and host tropism studies. It is easily propagated in a number of cell lines, including adherent cells and suspension cultures, and minimal purification is required to provide a stock for in vivo and in vitro studies. Curr. Protoc. Microbiol. 17:14A.1.1‐14A.1.20. © 2010 by John Wiley & Sons, Inc.

Keywords: poxvirus; myxoma virus; sucrose gradient purification; titration

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Roller Bottle Propagation of MYXV
  • Alternate Protocol 1: Propagation of Fluorescent Protein‐Tagged MYXV in Spinner Flasks
  • Alternate Protocol 2: Small‐Scale MYXV Amplification and Crude Virus Stock Purification
  • Basic Protocol 2: Purification of MYXV Using a Sucrose Cushion or Gradient
  • Basic Protocol 3: Quantification of Purified MYXV by Foci Forming Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Roller Bottle Propagation of MYXV

  Materials
  • Complete minimal essential medium (MEM; see recipe), 37°C
  • CV‐1 cells (ATCC #CCL‐70), BSC‐40, or BGMK cells grown to confluence in 150‐cm2 tissue culture flasks
  • Phosphate‐buffered saline (PBS; see recipe), 37°C
  • 1× trypsin/EDTA/PBS (see recipe), 37°C
  • 1 M HEPES, pH 7.4 (see recipe)
  • Concentrated MYXV stock
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • Expanded surface roller bottles (Fisher, cat. no. 06‐419‐9)
  • 37°C roller bottle incubator or other incubator that will accommodate a small roller device
  • Roller device that will fit in 37°C incubator
  • 50‐ml conical centrifuge tubes
  • Refrigerated low‐speed table‐top centrifuge (e.g., Sorvall Legend RT, Beckman GS‐6R, or equivalent)
  • Inverted tissue culture microscope
  • Fluorescent microscope

Alternate Protocol 1: Propagation of Fluorescent Protein‐Tagged MYXV in Spinner Flasks

  • BHK‐21 cells (ATCC #CCL‐10)
  • Complete Dulbecco's modified Eagle medium (complete DMEM; see recipe)
  • Complete Iscove's modified Dulbecco's medium (complete IMDM; see recipe)
  • MYXV tagged with a fluorescent protein
  • 10‐cm tissue culture plates
  • 37°C, 5% CO 2 incubator
  • 37°C water bath
  • 125‐ and 500‐ml spinner flasks (Bellco µ‐Carrier spinner flasks)
  • Disposable sterile pipets
  • Magnetic stir plate
  • 60‐mm tissue culture dishes or 6‐well culture dishes
  • 500‐ml centrifuge bottles
  • Sorvall SLA‐3000 rotor or equivalent
  • High‐speed centrifuge (e.g., Sorvall RC‐6 or equivalent)

Alternate Protocol 2: Small‐Scale MYXV Amplification and Crude Virus Stock Purification

  • BGMK cells
  • Swelling buffer (see recipe)
  • 150‐cm2 flasks
  • Platform rocker
  • Sterile cell scrapers
  • 40‐ml Dounce homogenizer with a loose pestle (autoclaved)
  • 50‐ml Oak Ridge tubes (Fisher, cat. no. NC9472311)
  • Sorvall SA600 rotor or equivalent
  • Cup sonicator, optional
  • 1.5‐ or 2‐ml cryotubes
CAUTION: All procedures should be performed in a Class II Biosafety cabinet, and liquid waste inactivated with BioClean, bleach, or other appropriate disinfectant to a final concentration of 10%. Autoclave all plasticware before disposing.NOTE: BGMK cells are not commercially available. The cells were a gift from Dr. Samuel Dales (University of Western Ontario, London, Ontario, Canada), who obtained them from SUNY Buffalo, where the cell line originated (Barron et al., ).NOTE: Perform all steps using aseptic technique, and assure that all reagents and supplies are sterile.

Basic Protocol 2: Purification of MYXV Using a Sucrose Cushion or Gradient

  Materials
  • Frozen suspension from harvested cells and virus (see protocol 1 and protocol 2)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A), 4°C
  • 24%, 28%, 32%, 36%, and 40% (w/v) sucrose in 10 mM Tris⋅Cl, pH 8.0 (see recipe): prepare the day before use and store overnight at 4°C
  • 37°C water bath
  • Cup sonicator
  • 40‐ml Dounce homogenizer with loose pestle (autoclaved)
  • 50‐ml conical centrifuge tubes
  • Refrigerated low‐speed table‐top centrifuge
  • Sterile individually wrapped pipets
  • 40‐ml Ultraclear ultracentrifuge tubes for SW28 rotor (Beckman Coulter, cat. no. 344058)
  • Ultracentrifuge with Beckman SW28 and SW41 rotors or equivalents
  • 2‐ml cryotubes
  • 12‐ml Ultraclear ultracentrifuge tubes for SW41 rotor (Beckman Coulter, cat. no. 344059)
  • Sterile Pasteur pipets
  • Spectrophotometer

Basic Protocol 3: Quantification of Purified MYXV by Foci Forming Assay

  Materials
  • CV‐1 cells (ATCC #CCL‐70) grown to near confluency in 75‐cm2 tissue culture flasks
  • Complete MEM medium (see recipe)
  • MYXV having the reporter gene lac Z (vMyxlac)
  • Crystal violet solution (see recipe)
  • PBS (see recipe), cold
  • Neutral‐buffered formalin (see recipe)
  • X‐gal staining solution (see recipe)
  • 6‐well tissue culture dishes
  • Inverted tissue culture microscope
  • 37°C, 5% CO 2 incubator
  • Cup sonicator, optional
  • Platform rocker, optional
  • Fluorescent microscope
CAUTION: All procedures should be performed in a Class II biosafety cabinet, and liquid waste inactivated with BioClean, bleach, or other appropriate disinfectant to a final concentration of 10%. Autoclave all plasticware before disposing.CAUTION: Dispose of all chemicals in the appropriate manner.NOTE: All tagged MYXV constructs were made in the McFadden laboratory.NOTE: Perform all steps using aseptic technique, and assure that all reagents and supplies are sterile.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Barron, A.L., Olshevsky, C., and Cohen, M.M. 1970. Characteristics of the BGM line of cells from African green monkey kidney. Arch. Gesamte Virusforsch. 32:389‐392.
   Cameron, C., Hota‐Mitchell, S., Chen, L., Barrett, J., Cao, J.X., Macaulay, C., Willer, D., and McFadden, G. 1999. The complete DNA sequence of myxoma virus. Virology 264:298‐318.
   Fenner, F. and Ratcliffe, F.N. 1965. Myxomatosis. Cambridge University Press, Cambridge, U.K.
   Johnston, J. and McFadden, G. 2003. Poxvirus immunomodulation strategies: Current perspectives. J. Virol. 77:6093‐6100.
   Kerr, P. and McFadden, G. 2002. Immune responses to myxoma virus. Viral Immunol. 15:229‐246.
   Kim, M., Madlambayan, G.J., Rahman, M.M., Smallwood, S.E., Meacham, A.M., Hosaka, K., Scott, E.W., Cogle, C.R., and McFadden, G. 2009. Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoietic stem and progenitor cells. Leukemia 23:2313‐2317.
   Kotwal, G.J. and Abrahams, M‐R. 2004. Growing poxviruses and determining virus titer. In Methods in Molecular Biology, Vol. 269: Vaccinia Virus and Poxvirology Methods and Protocols (S.N. Isaacs, ed.) pp. 101‐112. Humana Press, Totowa, New Jersey.
   Liu, L., Lalani, A., Dai, E., Seet, B, Macauley, C., Singh, R., Fan, L., McFadden, G., and Lucas, A. 2000. The viral anti‐inflammatory chemokine‐binding protein M‐T7 reduces intimal hyperplasia after vascular injury. J. Clin. Invest. 105:1613‐1621.
   Lucas, A. and McFadden, G. 2004. Secreted immunomodulatory viral proteins as novel biotherapeutics. J. Immunol. 173:4765‐4774.
   Lun, X., Yang, W., Alain, T., Shi, Z‐Q., Muzik, H., Barrett, J.W., McFadden, G., Bell, J., Hamilton, M.G., Senger, D.L., and Forsyth, P.A. 2005. Myxoma virus is a novel oncolytic virus with significant antitumor activity against experimental human gliomas. Cancer Res. 65:9982‐9990.
   McFadden, G. 2005. Poxvirus tropism. Nat. Rev. Microbiol. 3:201‐213.
   Regnery, D.C. 1971. The epidemic potential of Brazilian myxoma virus (Lausanne strain) for three species of North American cottontails. Am. J. Epidemiol. 94:514‐519.
   Silvers, L., Barnard, D., Knowlton, F., Inglis, B., Labudovic, A., Holland, M.K., Janssens, P.A., van Leeuwen, B.H., and Kerr, P.J. 2010. Host‐specificity of myxoma virus: Pathogenesis of South American and North American strains of myxoma virus in two North American lagomorph species. Vet. Microbiol. 141:289‐300.
   Stanford, M.M. and McFadden, G. 2007. Myxoma virus and oncolytic virotherapy: A new biological weapon in the war against cancer. Expert Opin. Biol. Ther. 7:1415‐1425.
   Stanford, M.M., Barrett, J.W., Nazarian, S.H., Werden, S., and McFadden, G. 2007a. Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells. J. Virol. 81:1251‐1260.
   Stanford, M.M., Werden, S.J., and McFadden, G. 2007b. Myxoma virus in the European rabbit: Interactions between the virus and its susceptible host. Vet. Res. 38:299‐318.
   Sypula, J., Wang, F., Ma, Y., Bell, J., and McFadden, G. 2004. Myxoma virus tropism in human tumor cells. Gene Ther. Mol. Biol. 8:103‐114.
Internet Resources
   http://www.cdc.gov/od/OHS/biosfty/bmbl5/BMBL_5th_Edition.pdf
  Website for U.S. Department of Health and Human Services' Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library