Human Cytomegalovirus: Propagation, Quantification, and Storage

William J. Britt1

1 University of Alabama School of Medicine, Birmingham, Alabama
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14E.3
DOI:  10.1002/9780471729259.mc14e03s18
Online Posting Date:  August, 2010
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Abstract

Human cytomegalovirus (HCMV) is the largest and perhaps the most structurally complex member of the family of human herpesviruses. It is the prototypic virus of the β‐herpesvirus subfamily. As with other cytomegaloviruses, HCMV is exquisitely species specific and undergoes lytic replication only in cells of human origin. In addition, its replication is limited almost entirely to primary cells and a limited number of transformed cell lines. Together with its prolonged replicative cycle of ∼48 hr, the propagation and quantification of HCMV can present technical challenges. In this brief set of protocols, the propagation of laboratory strains of HCMV and their quantitation is described. In a third series of protocols, the concentration and gradient purification of HCMV for more specialized downstream applications is described. Curr. Protoc. Microbiol. 18:14E.3.1‐14E.3.17. © 2010 by John Wiley & Sons, Inc.

Keywords: human cytomegalovirus; virus propagation; virus quantification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation and Preparation of Virus Stocks of Laboratory Strains of HCMV
  • Basic Protocol 2: Rapid Preparation of Viral Stocks from Supernatant and Virus‐Infected Cells
  • Alternate Protocol 1: Preparation of Stocks from Supernatant Virus
  • Alternate Protocol 2: Preparation of Virus Stocks from Supernatant Virus and Infected Cells
  • Basic Protocol 3: Quantification of HCMV Infectivity by Assay of Plaque‐Forming Units
  • Alternate Protocol 3: Titration of Infectivity Using Immunological Detection of Immediate‐Early Proteins
  • Basic Protocol 4: Preparation of Concentrated Virus by Centrifugation
  • Basic Protocol 5: Sorbitol Density Gradient Purification of HCMV
  • Alternate Protocol 4: Sucrose Density Gradient Purification of HCMV
  • Alternate Protocol 5: Glycerol/Tartrate Density Gradient Purification of HCMV
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation and Preparation of Virus Stocks of Laboratory Strains of HCMV

  Materials
  • 150‐cm2 flask of primary human fibroblasts at ∼80% confluency of the attached cells (∼1 × 107 cells; see Takashima, )
  • Supplemented Dulbecco's modified Eagles medium (see recipe)
  • HCMV seed virus: AD169 (ATCC VR58); Towne (ATCC VR977)
  • Platform rocker
  • 37°C humidified CO 2 incubator
NOTE: Cell lines for virus propagation: Primary human dermal fibroblasts derived from skin biopsies or from discarded human foreskin can be prepared in house. Alternatively, MRC5 human fibroblasts can be obtained from ATCC (CCL 171) or life‐extended human fibroblasts (telomerized primary human fibroblasts) can be obtained commercially or from the ATCC (BJ 5TA ATCC CRL‐4001).

Basic Protocol 2: Rapid Preparation of Viral Stocks from Supernatant and Virus‐Infected Cells

  Materials
  • Fetal bovine serum (FBS; Hyclone)
  • Virus culture ( protocol 1)
  • Cell scraper, sterile
  • 1.2‐ml cryovials
  • Microcentrifuge

Alternate Protocol 1: Preparation of Stocks from Supernatant Virus

  Materials
  • Virus culture ( protocol 1)
  • Sterile milk (see recipe)
  • 50‐ml conical centrifuge tubes
  • Centrifuge
  • 1.2‐ml cryovials

Alternate Protocol 2: Preparation of Virus Stocks from Supernatant Virus and Infected Cells

  Materials
  • Virus culture ( protocol 1)
  • Fetal bovine serum or sterile milk (see recipe)
  • Cell scraper, sterile
  • 50‐ml conical centrifuge tubes, sterile
  • Microtipped sonicator
  • 1.2‐ml cryovials
  • Centrifuge

Basic Protocol 3: Quantification of HCMV Infectivity by Assay of Plaque‐Forming Units

  Materials
  • 150‐cm2 flask of primary human fibroblasts at ∼80% confluency of the attached cells (∼1 × 107 cells; see Takashima, )
  • Viral stock (see protocol 2, protocol 3, or protocol 4)
  • Supplemented DMEM (see recipe)
  • Low‐melting‐point agarose
  • 2% formaldehyde in distilled water
  • 0.02% methylene blue in distilled water
  • 24‐well sterile tissue culture plate
  • Multichannel pipet (12 channels)
  • 5‐ml polypropylene tubes or 24‐well plates
  • 37°C, 5% CO 2 incubator
  • Inverted fluorescence microscope with emission 540‐nm filter
  • Aspirator

Alternate Protocol 3: Titration of Infectivity Using Immunological Detection of Immediate‐Early Proteins

  • Dulbecco's phosphate‐buffered saline (D‐PBS; see recipe)
  • 95% (v/v) ethanol
  • Anti‐immediate‐early 1 gene monoclonal antibodies (Millipore or other suppliers)
  • FITC goat anti–mouse IgG (Invitrogen or other suppliers)
  • Evans blue (Sigma, cat. no. E‐2129)
  • Glycerol
  • 96‐well flat‐bottomed tissue culture plates
  • Inverted epifluorescence microscope for viewing 96‐well plates (e.g., Zeiss model Axiovert 40 CFL with filter set to view fluoroscein or other fluorochromes)

Basic Protocol 4: Preparation of Concentrated Virus by Centrifugation

  Materials
  • 150‐cm2 flasks containing virus‐infected cells ( protocol 1)
  • Tris‐buffered saline (TN, 0.05 M Tris, 0.10 M NaCl, pH 7.4)
  • Sucrose solutions (20% w/v in TN; 50% w/v in TN)
  • Sterile 50‐ml conical centrifuge tubes
  • Centrifuge
  • 25 × 89–mm polycarbonate ultracentrifuge tubes
  • Glass or polyallomer tubes
  • Pasteur pipets
  • Ultracentrifuge (e.g., Beckman) and rotors (e.g., SW28)
  • Refrigerated high‐speed centrifuge (e.g., Beckman Avanti)

Basic Protocol 5: Sorbitol Density Gradient Purification of HCMV

  Materials
  • Concentrated virus solution (see protocol 7)
  • D‐Sorbitol solutions (20% w/v in TN; 70% w/v in TN)
  • Tris‐buffered saline (TN; see recipe)
  • Gradient maker with mixing chamber and reservoir (simple 2‐chamber gradient makers are available from number of manufacturers but can also be fabricated in‐house)
  • 14 × 89–mm polycarbonate tubes
  • Ultracentrifuge (e.g., Beckman) and rotors (e.g., SW41)
  • 3‐ml disposable plastic syringe equipped with a 20‐G 1‐in. needle
  • Vertical light source

Alternate Protocol 4: Sucrose Density Gradient Purification of HCMV

  • Potassium tartrate solution (15% w/v in TN; 35% w/v in TN)
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Figures

Videos

Literature Cited

Literature Cited
   Andreoni, M., Faircloth, M., Vugler, L., and Britt, W.J. 1989. A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus. J. Virol. Methods 23:157‐167.
   Boppana, S.B., Smith, R., Stagno, S., and Britt, W.J. 1992. Evaluation of a microtiter plate fluorescent antibody assay for rapid detection of human cytomegalovirus infections. J. Clin. Microbiol. 30:721‐723.
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