Epstein‐Barr Virus (EBV): Infection, Propagation, Quantitation, and Storage

Ke Lan1, Subhash C. Verma1, Masanao Murakami1, Bharat Bajaj1, Erle S. Robertson1

1 University of Pennsylvania Medical School, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14E.2
DOI:  10.1002/9780471729259.mc14e02s6
Online Posting Date:  August, 2007
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Abstract

Epstein‐Barr virus (EBV) was first reported as the etiological agent of Burkitt's lymphoma in 1964. Since then, EBV has also been associated with nasopharyngeal carcinoma, which is highly prevalent in Southeast Asia, as well as infectious mononucleosis, complications of AIDS, and transplant‐related B cell lymphomas. This virus has further been linked with T cell lymphomas and Hodgkin's disease, establishing the concept of a wide spectrum of EBV‐associated malignant disorders. So far, there are a number of EBV‐infected cell lines established that can be induced for production of infectious viral progeny and that facilitate the study of the mechanism of EBV‐related infection, transformation, and oncogenesis. This unit describes procedures for the preparation of EBV virion particles and in vitro infection of cells with EBV. In addition, procedures for quantitation and storage of the virus are provided. Curr. Protoc. Microbiol. 6:14E.2.1‐14E.2.21. © 2007 by John Wiley & Sons, Inc.

Keywords: Epstein Barr virus; induction; infection; quantitation; B lymphocyte

     
 
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Table of Contents

  • Introduction
  • Strategies for Induction of EBV Replication
  • Basic Protocol 1: Induction of EBV Replication Using Ig Cross‐Linking Agents
  • Basic Protocol 2: Induction of EBV Replication Using BZLF1 from a Heterologous Promoter
  • Basic Protocol 3: Induction of EBV Replication Using Phorbol Ester (12‐O‐Tetradecanoyl‐Phorbol‐13‐Acetate; TPA)
  • Basic Protocol 4: Induction of EBV Replication Using a Combination of TPA and Sodium Butyrate
  • Isolating EBV
  • Basic Protocol 5: Harvesting of EBV from Cell Supernatant
  • Basic Protocol 6: Concentration of EBV by Ultracentrifugation
  • Infecting Cells with EBV
  • Basic Protocol 7: Infection of Human Cells with EBV
  • Basic Protocol 8: Microcell‐Mediated EBV Genome Transfer to Mouse Cells
  • Quantitation of EBV
  • Basic Protocol 9: Quantitation of EBV Virions by Quantitative PCR Using DNA of Known Copy Number as Standard
  • Alternate Protocol 1: Quantitation of EBV Virions by Quantitative PCR via Comparison to Cell Line with Known Viral Copies
  • Long‐Term Storage of EBV
  • Basic Protocol 10: Freezing EBV‐Infected Cells
  • Basic Protocol 11: Thawing EBV‐Infected Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of EBV Replication Using Ig Cross‐Linking Agents

  Materials
  • EBV‐positive B cells (see protocol 7): Akata cells infected with GFP‐tagged EBV are recommended (Kanda et al., ; to obtain cells, contact Dr. Kanda at )
  • Complete RPMI medium containing 10% FBS (see recipe)
  • Anti‐IgG antibody
  • Centrifuge with swinging‐bucket rotor
  • Additional reagents and equipment for harvesting EBV from cell supernatant ( protocol 5)

Basic Protocol 2: Induction of EBV Replication Using BZLF1 from a Heterologous Promoter

  Materials
  • EBV‐positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2 (generated in Dr. Erle Robertson's laboratory; )
  • Complete RPMI medium containing 10% FBS (see recipe) and serum‐free RPMI 1640 medium (e.g., Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1µg/ml BZLF1 expression vector in TE buffer, pH 8.0 ( appendix 2A) or distilled H 2O
  • Centrifuge with swinging‐bucket rotor
  • 0.4‐cm‐gap electroporation cuvette
  • Gene pulser II system (Bio‐Rad)
  • Additional reagents and equipment for harvesting EBV from cell supernatant ( protocol 5)
NOTE: B95.8, LCL1, and LCL2 are all EBV‐positive B cells lines that can be used as a resources for production of the virus, with no significant difference between them.

Basic Protocol 3: Induction of EBV Replication Using Phorbol Ester (12‐O‐Tetradecanoyl‐Phorbol‐13‐Acetate; TPA)

  Materials
  • EBV‐positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2 (generated in Dr. Erle Robertson's laboratory; )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete RPMI medium containing 10% FBS (see recipe)
  • 2000× (40 µg/µg/ml) 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA; Sigma), sterilized by filtration through 0.22‐µm membrane
  • Centrifuge with swinging‐bucket rotor
  • Additional reagents and equipment for harvesting EBV from cell supernatant ( protocol 5)
NOTE: B95.8, LCL1, and LCL2 are all EBV‐positive B cells lines that can be used as a resources for production of the virus, with no significant difference between them.

Basic Protocol 4: Induction of EBV Replication Using a Combination of TPA and Sodium Butyrate

  Materials
  • EBV‐positive B cell line: B95.8 (available from ATCC) or LCL1 or LCL2 (generated in Dr. Erle Robertson's laboratory; )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete RPMI 1640 medium containing 10% FBS (see recipe)
  • 2000× (40 µg/µg/ml) 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA; Sigma), sterilized by filtration through 0.22‐µm membrane
  • 3 M sodium butyrate (SB), sterilized by filtration through 0.22‐µm membrane
  • Centrifuge with swinging‐bucket rotor
  • Additional reagents and equipment for harvesting EBV from cell supernatant ( protocol 5)
NOTE: B95.8, LCL1, and LCL2 are all EBV‐positive B cells lines that can be used as a resources for production of the virus, with no significant difference between them.

Basic Protocol 5: Harvesting of EBV from Cell Supernatant

  Materials
  • Cells induced for EBV replication (Basic Protocols protocol 11 to protocol 44)
  • Dry ice
  • Isopropanol
  • Centrifuge with swinging‐bucket rotor
  • 15‐ml conical centrifuge tubes
  • Container suitable for resisting extremely cold temperatures
  • 0.45‐µm syringe filter

Basic Protocol 6: Concentration of EBV by Ultracentrifugation

  Materials
  • 70% (v/v) ethanol
  • EBV‐containing supernatant ( protocol 5)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10‐ml ultracentrifuge tube
  • UV light source for sterilization
  • Ultracentrifuge with swinging‐bucket rotor (Fig. C)
  • Refrigerated centrifuge with fixed‐angle rotor accommodating 1.7‐ml microcentrifuge tubes

Basic Protocol 7: Infection of Human Cells with EBV

  Materials
  • Human B cell lines (suspension), human epithelial cell lines (adherent), or primary human B cells ( appendix 4C)
  • Complete RPMI medium containing 10% FBS (see recipe)
  • Supernatant containing EBV infectious virions ( protocol 5 or protocol 66)
  • 1× trypsin/EDTA solution (e.g., Invitrogen)
  • Complete DMEM medium containing 10% FBS (see recipe)
  • 15‐ml conical polypropylene centrifuge tubes
  • Centrifuge with swinging‐bucket rotor
  • 75‐cm2 tissue culture flasks or 100‐mm tissue culture plates
  • 96‐well tissue culture plate (optional; for producing lymphoblastoid cell line)
NOTE: Medium containing 10% FBS is not always necessary; lower serum concentrations have also been shown to work well for a number of cell lines.

Basic Protocol 8: Microcell‐Mediated EBV Genome Transfer to Mouse Cells

  Materials
  • EBV‐infected ( protocol 7) and EBV‐negative human lymphoid cell lines
  • Serum‐free RPMI 1640 medium (e.g., Invitrogen)
  • EBV‐hyg (hygromycin resistance gene–tagged EBV) or EBV‐neo (neomycin resistance gene–tagged EBV)
  • Lipofectamine 2000 transfection reagent (Invitrogen)
  • Fetal bovine serum (FBS; appendix 2A), heat inactivated
  • EBV‐A9 selection medium A (see recipe)
  • Colcemid (demecolcine; e.g., Invitrogen or Sigma)
  • Percoll microcell gradient mixture (see recipe)
  • Serum‐free DMEM medium (e.g., Invitrogen) containing 100 µg/ml phytohemagglutinin (PHA)
  • A9 cells (mouse B lymphocyte cell line used here as recipient cells; ATCC #CRL‐1811) grown as monolayer in 100‐mm culture dish
  • 47% (w/v) PEG (mol. wt. 8000)
  • Serum‐free DMEM medium (e.g., Invitrogen)
  • Growth medium for A9 cells: complete DMEM medium containing 10% FBS (see recipe)
  • 1× trypsin/EDTA solution (e.g., Invitrogen)
  • EBV‐A9 selection medium B (see recipe)
  • 6‐well tissue culture plates
  • 15‐ and 50‐ml centrifuge tubes
  • Centrifuge and plate carrier
  • 8‐, 5‐, and 3‐µm pore‐size Nucleopore polycarbonate filters (Whatman)
  • 25‐cm2 tissue culture flasks
  • 100‐mm tissue culture plates

Basic Protocol 9: Quantitation of EBV Virions by Quantitative PCR Using DNA of Known Copy Number as Standard

  Materials
  • Concentrated EBV virion suspension ( protocol 6)
  • 30% and 60% (w/v) sucrose solutions
  • 20% and 35% (w/v) Nycodenz (e.g., Sigma)
  • 0.5× and 1× phosphate‐buffered saline (PBS; see appendix 2A for 1× concentration)
  • Lysis buffer (see recipe)
  • 12:12:1 (v/v/v) phenol:chloroform:isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol, ice‐cold
  • Primers for quantitative PCR (Table 14.2.1)
  • DNA of known copy number as standard for quantitative PCR (Bookout et al., )
  • 15‐ml ultracentrifuge tubes
  • Gradient maker
  • Ultracentrifuge
  • 18.5‐G needles and syringes
  • MWCO 10,000 dialysis tubing, and dialysis clips
  • 60°C water bath
  • Additional reagents and equipment for quantitative PCR (Bookout et al., )
    Table 4.0.1   MaterialsPrimers Used For the Quantitation of EBV

    Regions Sense (5′‐3′) Antisense (5′‐3′)
    Bam C GCAGGGCTCGCAAAGTATAG TGCGGAAGTGACACCAAATA
    Bam E TACTGCCACCAGTACCACAACA GGCCGACATTCTCCAAGATAA
    EBNA2 CTCTGCCACCTGCAACACTA ATTTGGGGTGCTTTGATGAG
    LMP2 TGCAATTTGCCTAACATGGA TGGACATGAAGAGCACGAAG
    Bam W CCAGACAGCAGCCAATTGTC GGTAGAAGACCCCCTCTTAC

Alternate Protocol 1: Quantitation of EBV Virions by Quantitative PCR via Comparison to Cell Line with Known Viral Copies

  Materials
  • EBV‐infected or lymphoblastoid cells (see Basic Protocols protocol 77 and protocol 88)
  • Fetal bovine serum (FBS; appendix 2A)
  • EBV‐infected‐cell freezing medium (see recipe)
  • Cryotubes
  • Liquid nitrogen or ultracold freezer at –140°C

Basic Protocol 10: Freezing EBV‐Infected Cells

  Materials
  • Frozen EBV‐infected cells in cryotubes ( protocol 1)
  • Growth medium appropriate for cells used (see Reagents and Solutions)
  • Circular floating tube rack (Bioexpress, http://www.bioexpress.com; cat. no. R‐8011‐2)
  • 15‐ml conical polypropylene centrifuge tubes
  • Centrifuge
  • 25‐cm2 tissue culture flasks
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Figures

Videos

Literature Cited

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