Newcastle Disease Virus: Propagation, Quantification, and Storage

Lori W. McGinnes1, Homer Pantua1, Julie Reitter1, Trudy G. Morrison1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15F.2
DOI:  10.1002/9780471729259.mc15f02s01
Online Posting Date:  June, 2006
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Abstract

Newcastle disease virus (NDV) is a prototype paramyxovirus used to define basic steps in the life cycle of this family of viruses. NDV is also an ideal virus system for elucidating determinants of viral pathogenicity. Some strains of this virus are important agricultural pathogens that cause disease in poultry with a high mortality while other strains are avirulent and used for vaccines. Methods for preparation and titration of virus stocks are essential for all of these purposes. Procedures for growth and purification of NDV stocks in embryonated chicken eggs as well as in tissue culture cells are described. Use of embryonated chicken eggs to grow the virus is the superior method since infectious stocks of all strains of NDV result. Stocks of avirulent NDV prepared in tissue culture are noninfectious. Virus stocks are routinely titered using plaque assays or hemagglutination assays, both of which are described.

Keywords: Newcastle disease virus; paramyxovirus; embryonated eggs; virus purification; plaque assay

     
 
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Table of Contents

  • Basic Protocol 1: Plaque Assay for Newcastle Disease Virus
  • Basic Protocol 2: Preparation of Newcastle Disease Virus Zero Stocks
  • Basic Protocol 3: Growth of Virus in Embryonated Chicken Eggs
  • Basic Protocol 4: Purification of Virus from the Allantoic Fluid of Infected Eggs
  • Alternate Protocol 1: Growth and Purification of Virus Derived from Infection of Tissue Culture Cells
  • Basic Protocol 5: Titration of Virus Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Plaque Assay for Newcastle Disease Virus

  Materials
  • Avian tissue culture cells (e.g., ELL‐O chicken cells; ATCC #UMNSAH/DF‐1)
  • Virus stock (see Table 15.2.1; Southeast Poultry Research Laboratory, Athens, GA)
  • Tissue culture medium (e.g., DMEM with supplements; see recipe)
  • NDV overlay medium with or without trypsin, 46°C (see reciperecipes)
  • Methanol (optional)
  • Giemsa stain (Sigma; optional)
    Table 5.0.1   MaterialsRepresentative NDV Strains

    NDV strain a F protein cleavage site b Egg virulence c
    B1 Hitchner avirulent lentogenic
    La Sota avirulent lentogenic
    D26/76 avirulent lentogenic
    Queensland avirulent lentogenic
    Ulster avirulent lentogenic
    Roakin virulent mesogenic
    L virulent mesogenic
    Beaudette C virulent velogenic
    Italien virulent velogenic
    Texas G. B. virulent velogenic
    Hertz virulent velogenic
    Australia‐Victoria virulent velogenic
    Miyadera virulent velogenic

     aSee Pedersen, et al. (2004) for more recent isolates.
     bVirulent: F protein cleavage site is RXKR or RXRR; avirulent, F protein cleavage site is RQGR (Glickman et al., ; Toyoda et al., , )
     cEgg virulence classification based on approximate time required to kill chicken embryos: lentogenic, >90 hr; mesogenic, 60 to 90 hr; velogenic, 40 to 60 hr (Hanson and Brandly, ; Waterson et al., ; Toyoda et al., ; Alexander, ).
  • 35‐ or 60‐mm tissue culture plates
  • Pipets
  • 46°C water bath
NOTE: All culture incubations are performed in a humidified 37.5°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Preparation of Newcastle Disease Virus Zero Stocks

  Materials
  • Viral plaque ( protocol 1)
  • Hanks' balanced salt solution (HBSS; appendix 2A or Gibco 24020; contains calcium, magnesium, and phenol red)
  • Pasteur pipets
  • 1.5‐ml microcentrifuge tubes (polypropylene)
  • Microcentrifuge (Eppendorf or comparable)
  • 15‐ml polypropylene centrifuge tubes (Corning or comparable)
  • Sorvall centrifuge with an SS‐34 rotor (or comparable)
  • Additional reagents and equipment for growth of virus in eggs ( protocol 3, collection of allantoic fluid ( protocol 4), and obtaining plaques ( protocol 1)

Basic Protocol 3: Growth of Virus in Embryonated Chicken Eggs

  Materials
  • <10‐day‐old special pathogen free (SPF) eggs (e.g., Charles River/SPAFAS)
  • Zero stock ( protocol 2)
  • Hanks' balanced salt solution (HBSS; appendix 2A or Gibco 24020; contains calcium, magnesium, and phenol red) containing 2% (v/v) heat‐inactivated FBS ( appendix 2A)
  • 70% ethanol
  • Humidified egg incubator (e.g., Lyon Electric)
  • Egg candler (e.g., Lyon Electric)
  • 16‐G, 10‐mm needle
  • 1‐ml tuberculin syringe with an attached 26‐G, 10‐mm needle (e.g., Becton Dickinson)
  • Nail polish or wax

Basic Protocol 4: Purification of Virus from the Allantoic Fluid of Infected Eggs

  Materials
  • Infected eggs ( protocol 3)
  • 70% ethanol
  • Hanks' balanced salt solution (HBSS; appendix 2A or Gibco 24020; contains calcium, magnesium, and phenol red)
  • 70% ethanol/1% (w/v) SDS
  • HBSS ( appendix 2A or Gibco 24020) containing 2% heat‐inactivated FBS ( appendix 2A)
  • 20%, 25%, 35%, 45%, 55%, and 65% (w/v) sucrose in standard buffer (see below for standard buffer)
  • Standard buffer: 0.1 M NaCl/0.01 M Tris·Cl, pH 7.4 ( appendix 2A)/2 mM EDTA
  • Forceps
  • 10‐ml syringe with a 16‐G needle
  • Microspoon‐end spatula (e.g., VWR)
  • 250‐ml bottles for GSA rotor (or comparable)
  • Sorvall centrifuge (or comparable) with GSA rotor
  • Beckman Type 19 bottles
  • Deldrin cap assemblies (or comparable): sterilize by rinsing with 70% ethanol and exposing to UV light for 20 hr
  • Beckman ultracentrifuge with type 19 rotor
  • 10‐ml glass Dounce homogenizer with a tight‐fitting pestle
  • 30‐ml Sorvall centrifuge tube
  • Sorval SS‐34 rotor
  • Beckman SW28.1 rotor with large buckets and small buckets
  • 17‐ and 30‐ml Beckman ultracentrifuge tubes (or comparable)
  • Ring stand with clamps
  • 10‐ml syringe with a 21‐G needle
  • 1‐ or 2‐ml cryovials
  • −80°C freezer
NOTE: Precool all centrifuges and rotors and chill all solutions and glassware used for purification of virus.NOTE: The following steps are for ten dozen infected eggs.

Alternate Protocol 1: Growth and Purification of Virus Derived from Infection of Tissue Culture Cells

  Materials
  • Avian tissue culture cells (e.g., ELL‐O chicken cells; ATCC #UMNSAH/DF‐1)
  • Tissue culture medium (e.g., DMEM with supplements; see recipe)
  • Virus zero stock ( protocol 2), titered
  • 100‐mm tissue culture plates (avian cells)
  • 37.5°C, 5% CO 2 tissue culture incubator, humidified
  • Additional reagents and equipment for virus concentration and sucrose gradient centrifugation (see protocol 4)

Basic Protocol 5: Titration of Virus Stocks

  Materials
  • Red blood cells (avian alsever or guinea pig alsever; BioLink)
  • PBS, ice cold ( appendix 2A)
  • 1.5 ml microcentrifuge tubes
  • Microcentrifuge
  • Hemacytometer
  • Microscope
  • Microtiter plates (96‐well, round bottom)
NOTE: Maintain all solutions on ice.
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Figures

Videos

Literature Cited

Literature Cited
   Alexander, D.J. 1997. Newcastle disease and other avian paramyxoviruses. In Diseases of Poultry (B.W. Cainek, H.J. Barnes, C.W. Beard, L.R. McDougald, and L.J. Saif, eds.) pp. 541‐570. Iowa State University Press, Ames, Iowa.
   Beard, C.W. and Hanson, R.P. 1984. Newcastle disease. In Disease of Poultry (M.S. Hofstad, ed.) pp. 452‐470. Iowa State University Press, Ames, Iowa.
   Garten, W., Berk, W., Nagai, R., Rott, R., and Klenk, H.D. 1980. Mutational changes of the protease susceptibility of glycoprotein F of Newcastle disease virus: Effects on pathogenicity. J. Gen. Virol. 50:135‐147.
   Glickman, R.L., Syddall, R.J., Sheehan, R.M., and Bratt, M.A. 1988. Quantitative basic residue requirements in the cleavage activation site of the fusion glycoprotein as a determinant of virulence of Newcastle disease virus. J. Virol. 62:354‐356.
   Gotoh, B., Ogasawara, T., Toyoda, T., Inocencio, N.M., Hamaguchi, M., and Nagai, Y. 1990. An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo. EMBO J. 9:4189‐4195.
   Gotoh, B., Ohnishi, Y., Inocencio, N.M., Esaki, E., Nakayama, K., Barr, P.J., Thomas, G., and Nagai, Y. 1992. Mammalian subtilisin‐related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: Superiority of furin/PACE to PC2 or PC1/PC3. J. Virol. 66:6391‐6397.
   Hanson, R.P. and Brandly, C.A. 1955. Identification of vaccine strains of Newcastle disease virus. Science 122:156‐157.
   Lamb, R.A. and Kolakofsky, D. 2001. Paramyxoviridae: The viruses and their replication. In Fields Virology, Vol. 1, 3rd ed., (D.M. Knipe and P.M. Howley, eds.) pp. 1305‐1340. Lippincott Williams & Wilkins, Philadelphia.
   Morrison, T.G. 2003. Structure and function of a paramyxovirus fusion protein. Biochim. Biophys. Acta 1614:73‐84.
   Nagai, Y. 1993. Protease‐dependent virus tropism and pathogenicity. Trends Microbiol. 1:81‐87.
   Nagai, Y. and Klenk, H.D. 1977. Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage. Virology 77:125‐134.
   Nagai, Y., Klenk, H.D., and Rott, R. 1976. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:494‐508.
   Nagai, Y., Hamaguchi, M., and Toyoda, T. 1989. Molecular biology of Newcastle disease virus. Prog. Vet. Microbiol. Immunol. 5:16‐64.
   Pedersen, J.C., Senne, D.A., Woolcock, P.R., Kinde, H., King, D.J., Wise, M.G., Panigrahy, B., and Seale, B.S. 2004. Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002‐2003 outbreak in California and other recent outbreaks in North America. J. Clin. Microbiol. 42:2329‐2334.
   Rott, R. and Klenk, H.D. 1988. Molecular basis of infectivity and pathogenicity of Newcastle disease virus. In Newcastle Disease (D.J. Alexander, ed.) pp. 98‐112. Kluwer Academic Publishers, Boston.
   Toyoda, T., Sakaguchi, T., Imai, K., Inocencio, N.M., Gotoh, G., Hamaguchi, M., and Nagai, Y. 1987. Structural comparison of the cleavage‐activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus. Virology 158:242‐247.
   Toyoda, T., Sakaguchi, T., Hirota, H., Gotoh, B., Kuma, K., Miyata, T., and Nagai, Y. 1989. Newcastle disease virus evolution. II. Lack of gene recombination in generating virulent and avirulent strains.Virology 169:273‐82.
   Waterson, A.P., Pennington, T.H., and Allan, W.H. 1967. Virulence in Newcastle disease virus: A preliminary study. British Med. Bull. 23: 138‐143.
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