BART, BamHI rightward transcript; EBNA, Epstein-Barr nuclear antigen; LMP, latent membrane protein; snRNP, small nuclear ribonucleoprotein; ZEBRA, BZLF-1 EBV replication activator.

BART, BamHI rightward transcript; EBNA, Epstein-Barr nuclear antigen; LMP, latent membrane protein; snRNP, small nuclear ribonucleoprotein; ZEBRA, BZLF-1 EBV replication activator.

a Forward primers are given first, reverse (antisense) primers second, and internal oligonucleotide probes (oligonucleotide name starts with "p") as third for each target. Most primers are designed to be intron-flanking to prevent background genomic DNA amplification. For some targets, background DNA amplification will yield higher bands (indicated in column 4). The sequence of the probe for EBNA-2 transcripts is the same as for the EBNA-1 Y3 forward primer.

2. 10X PCR buffer: 50 mM Tris-HCl, 440 mM KCl, 12 mM MgCl2, pH 8.3.

3. dNTP stock solution containing 2 mM each of dATP, dTTP, dCTP, and dGTP.

4. Forward and reverse primers (see Table 1) in a concentration of 100 pmol/|L.

5. 5 U/|L, AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA).

6. PCR thermocycler (e.g., Applied Biosystems PE 9600).

2.4. Detection of PCR Products by Hybridization With Radiolabeled Internal Oligonucleotide Probes

2.4.1. Electrophoretic Separation of PCR Products and Blotting to Nylon

1. Agarose

2. TBE: 90 mM Tris, 80 mM boric acid, 2 mM EDTA, pH 8.0.

3. Electropheresis unit.

4. Nylon membrane, e.g., GeneScreen Plus (Nen Life Science, Boston, MA).

5. Blotting buffer: 0.4 M NaOH.

6. Capillary blotting setup.

7. Hybridization buffer: 0.5 M Na2HPO4, 7% sodium dodecyl sulfate [SDS], pH corrected to 7.2 with H3PO4.

8. Plastic tray with hermetic seal.

2.4.2. Radioactive 5'-End-Labeling of Oligonucleotides by T4 Polynucleotide Kinase

1. T4 polynucleotide kinase (New England Biolabs, Beverley, MA).

2. 10X kinase buffer (New England Biolabs).

3. [y-32P]ATP (370 MBq/mL; 10 mCi/ml; Amersham Bioscience, Piscataway, NJ).

4. Glass Pasteur pipet (diameter 5 mm).

5. Glass wool.

6. Sepharose G50 (Pharmacia Biotech, Uppsala, Sweden).

8. Water bath with agitation.

9. Wash buffer: 3X SSC/0.1% SDS (0.45 MNaCl, 0.045 MNa-citrate, 1% SDS, pH 7.0).

10. Geiger-Mülller counter, e.g., Mini Monitor Series 900 G-M tube (MiniInstruments, Burnham-on-Crouch, UK) or Liquid Scintillation Counter, e.g., TriCarb 1900 TR (Packard Bioscience, Boston, MA)

11. Film caset.

12. Photographic film, e.g., Kodak X-OMAT AR 3. Methods

3.1. RNA Isolation From Clinical Specimens

1. Cut 5-10 cryosections of 5 |M (depending on biopsy size) and immediately add 1 mL of RNA-Bee RNA Isolation Solvent. For isolated or cultured cells, add 0.2 mL RNA-Bee per million cells. Vortex thoroughly, and store on ice for 5 min (see Notes 4 and 5). For each experiment include a positive control, such as an EBV-

positive cell line with latency type 3 (e.g. JY, JC5, or Raji) or clinical specimens collectively expressing the EBV genes to be amplified.

2. Add 1/10th vol of chloroform. Vortex for 15 s, and store on ice for 5 min.

3. Centrifuge 1 mL of the suspension for 15 min at maximum speed in a standard high-speed benchtop centrifuge (12,000g).

4. Transfer the aqueous (colorless) phase to a new reaction tube, add an equal volume of isopropanol, and incubate on ice for 15 min (see Notes 6 and 7).

5. Centrifuge 250 |L of the isopropanol-RNA solution for 30 min at maximum speed in standard benchtop centrifuge (see Note 8)

6. Remove supernatant (see Note 9) and add 500 |L of cold (-20°C) 75% ethanol. Vortex briefly, and centrifuge for 5 min at maximum speed.

7. Remove supernatant and dry RNA pellet at room temperature (see Note 10).

3.2. EBV Gene-Specific Multiprimed cDNA Synthesis

1. Dissolve the RNA pellet in 5 |L of multi-RT-primer mix.

2. Incubate at 65°C for 5 min to denature secondary RNA structures. After incubation, immediately place the sample on ice. Centrifuge briefly after 3 min.

3. Add 15 |L of RT reaction mix to the RNA sample. The RT reaction mix contains per reaction (for preparation of RT master mix multiply volumes by the amount of samples to be analyzed): - 2 |L 10X RT buffer, 2 |L 100 mM DTT, 10.6 |L 2 mM dNTPs, 0.2 |L RNasin, and 0.2 |L AMV-RT (see Note 11).

4. Incubate at 42°C for 60 min for cDNA synthesis (see Note 12).

3.3. cDNA Amplification by PCR

1. After cDNA synthesis, supplement the RT reaction with 25 |L of RT reaction supplement buffer to obtain a volume large enough for the subsequent nine different PCR reactions.

2. Divide 5 |L aliquots of of supplemented RT reaction in nine PCR tubes.

3. Add 45 |L PCR mix, respectively, with forward and antisense primers for either of the following EBV transcripts:

a. EBNA-1 QK splice variants (primers EBNA-1 Q and EBNA- 1 K).

b. EBNA-1 Y3K splice variants (primers EBNA-1 Y3 and EBNA-1 K).

e. LMP-2a (primers LMP-2a1 and LMP-2ab2).

f. LMP-2b (primers LMP-2b1 and LMP-2ab2).

i. The cellular housekeeping gene U1A snRNP (primers U1A1 and U1A2).

The PCR mix contains per reaction (see Notes 13 and 14): 36.8 |L water, 5 |L 10X PCR buffer, 0.25 |L forward primer (100 pmol/|L), 0.25 |L reverse primer (100 pmol/|L), 2.5 |L 2 mM dNTPs, and 0.2 |L AmpliTaq DNA polymerase.

4. Cycle the samples in a PCR device using the following PCR program: 4 min at 95°C; 40 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; 7 min at 72°C, and finally a hold at 4°C (see Note 15).

3.4. Detection of PCR Products by Hybridization With Radiolabeled Internal Oligonucleotide Probes

3.4.1. Electrophoretic Separation of PCR Products and Blotting to Nylon

1. Separate PCR products and a molecular size marker (e.g., a 100-bp ladder or 2-log ladder available from New England Biolabs) by standard 1.5% agarose gel electrophoresis in TBE buffer for 1-2 h at approx 100 mA.

2. Transfer the PCR products from agarose gel to nylon by standard alkaline capillary blotting in blotting buffer.

3. Mark orientation of the samples on the nylon membrane.

4. Neutralize the nylon membrane by washing three times 5 min in 2X SSC.

5. Air-dry the nylon membrane.

6. Incubate the nylon membrane with 50 mL hybridization buffer in a fully sealed plastic tray placed in a water bath at 55°C for at least 15 min (see Note 16).

3.4.2. Radioactive 5'-End-Labeling of Oligonucleotides by T4 Polynucleotide Kinase and Detection of PCR Products by Autoradiography (see Note 17)

1. Label the internal oligonucleotides for detection of PCR products using the following reaction): 14 ||L water, 1 ||L olignucleotide probe (20 pmol/|L), 2 ||L 10X kinase buffer (New England Bio Labs), 1 |L T4 polynucleotide kinase (New England Bio Labs), and 2 |L [y-32P]ATP.

3. Pipet labeling reaction onto Sepharose G50 column (see Note 18) to separate the labeled oligonucleotide probe from free (nonincorporated) label by gel filtration.

4. Repeatedly pipet 200 | L fractions of TE onto the column and collect fractions of 200 | L.

5. Measure activity of the collected fractions by P-radiation monitor or scintillation counter. Gel filtration will yield two peaks of activity, the first peak containing the labeled oligonucleotide probes (activity >500 counts/s, typically fractions three to five) and the second peak containing the free label. Pool the fractions containing the oligonucleotide probe, and add to the nylon membrane in hybridization buffer (see Subheading 3.4., step 4).

6. Incubate for 16-20 h in a fully sealed plastic tray (max. 7 x 20 cm) in a water bath at 55°C with agitation (see Note 19).

7. Collect hybridization buffer containing the labeled probe for reuse (see Note 20). Wash three times by adding a small volume of wash buffer (covering the nylon membrane completely) and incubating for >15 min at 55°C using agitation. Measure activity of wash buffer after the last washing step. If this is still active, additional washing steps may be performed until no activity is detected.

Fig. 1. Autoradiograph showing relative sensitivity of multiprimed EBV RT-PCR analysis. A dilution series of 1-100,000 EBV-positive JY cells was made in a whole blood DNA background, originating from an EBV-negative donor. JY is a lymphoblas-toid cell line that expresses EBNA-1 mainly from the C/W promotor and shows limited ZEBRA expression in <1% of the cells. For most transcripts, RNA from at least one cell equivalent could be detected, except for EBNA-1 QK and ZEBRA RNA, which are expressed in only a minority of JY cells. Analytical sensitivity is at least 10 RNA, copies for each target (4).

Fig. 1. Autoradiograph showing relative sensitivity of multiprimed EBV RT-PCR analysis. A dilution series of 1-100,000 EBV-positive JY cells was made in a whole blood DNA background, originating from an EBV-negative donor. JY is a lymphoblas-toid cell line that expresses EBNA-1 mainly from the C/W promotor and shows limited ZEBRA expression in <1% of the cells. For most transcripts, RNA from at least one cell equivalent could be detected, except for EBNA-1 QK and ZEBRA RNA, which are expressed in only a minority of JY cells. Analytical sensitivity is at least 10 RNA, copies for each target (4).

8. Seal nylon membrane in plastic. First, carefully remove remaining liquid by rubbing the partially sealed membrane with force and using tissues to absorb excess liquid. Then seal the membrane completely and expose overnight at -80°C in a cas-sete to a X-ray film (e.g., Kodak safety Film AR O).

9. Develop the film in an X-ray developing machine or manually using photo developing solutions (e.g., Agfa). An example can be seen in Fig. 1.

4. Notes

1. Because RNases are omnipresent, it is essential to use RNase-free disposables such as reaction tubes and pipet tips. The use of filter tips is strongly recommended. Glassware should be sealed with aluminium foil and baked for >2 h at 180°C to degrade RNAses, which are resistant to autoclaving. Solutions and reagents should be ordered either RNase free or treated with diethyl pyrocarbonate (DEPC; add 0.1% [(v/v)] of DEPC and autoclave). Note that some chemicals, e.g., Tris-HCl are reactive with DEPC and should not be DEPC-treated. Aliquoting of all reagents including primers is recommended to avoid contamination with RNases or PCR products by carryover.

2. RNA-Bee should be stored in the dark at 2-8°C. It contains guanidium thiocyanate, which is an irritant, and phenol, which is toxic by ingestion, skin contact, and inhalation of vapor. Discard waste as appropriate, according to environmental safety guidelines.

3. For cDNA synthesis from the EBNA-1 QK and EBNA-1 Y3K splice variants, the same antisense primer (EBNA-1 K) is used. The same holds true for the LMP-2a and -2b (primer LMP-2ab2). Thus, a total of seven antisense primers is used for multiprimed cDNA synthesis of nine targets.

4. Because RNA is extremely sensitive to omnipresent RNases, precautions should be taken to protect RNA from enzymatic degradation. These include wearing gloves when handling RNA, opening tubes only if necessay for short periods, and cleaning lab benches with bleach, ethanol, or 0.1% SDS before starting the experiment.

5. For isolation of RNA direct from unfractionated whole blood, the RNA-Bee method is unsuited. For this we prefer the NucliSens® Nucleic Acid Isolation Method (BioMerieux, Boxtel, The Netherlands), which is a silica-based RNA extraction method efficiently removing substances putatively interfering with amplification in RT-PCR (10,11).

6. Make sure that no organic phase is removed. It is advised to leave a small amount of aqueous phase on top to avoid this.

7. The RNA/isopropanol solution can be stored long term at -80°C. Always keep on ice when pipeting the required volume from the RNA/isopropanol stock solution and return solution to -80°C immediately after use.

8. The amount of RNA/isopropanol to be used for RNA precipitation may be varied when preferred or according to availability of clinical material. For EBV RNA profiling in tissue biopsies, we routinely use the RNA equivalent of 2.5 cryosections (5 |im).

9. Place tubes in centrifuge with marked orientation, as the RNA pellet may not always be visible after precipitation. Be careful not to touch the pellet when discarding the supernatant.

10. When visible, the pellet may have a white appearance but should be glassy and transparent after drying. White pellets after drying indicate too much salt present. To avoid this, an additional washing step with 75% ethanol may be required. It is important not to let the RNA pellet dry completely, as this greatly decreases its solubility. Do not dry RNA by centrifugation under vacuum. Dissolve the RNA by passing the solution through a pipet tip and/or incubating for 10-15 min at 55-60°C. The final preparation of RNA has a A260/A280 ratio 1.6-1.9.

11. Store all RT reagents at -20°C. After thawing, keep reagents on ice. Make sure all reagents are completely dissolved before use, especially DTT, which easily precipitates at low temperatures.

12. cDNA may be stored for the long term at -20°C until used for amplification by PCR.

13. Prepare PCR mix on ice. Keep all reagents for PCR on ice after thawing to avoid nonspecific Taq DNA polymerase activity and primer-dimer formation. Store PCR reagents at -20°C.

14. To decrease the risk of DNA contamination of the PCR reactions and false positiv-ity, clean laboratory bench and pipets with 0.1 M HCl or 10% bleach and subsequently with water before starting the experiment. During the experiment open tubes only if necessary. Wear gloves when pipeting. Use filter tips for all pipeting. Use separate laboratories for preparation of PCR mixes, isolating DNA, and amplification. Aliquot all PCR reagents (12).

15. PCR products can be stored at 4°C for 1-2 d. For long-term storage, it is advised to use -20°C to avoid enzymatic PCR product degradation by 5'-exonuclease activity of Taq polymerase.

16. Nylon membranes can be placed on top of each other to a maximum of three, as long as they are completely covered in hybridization buffer and are able to move independently of each other.

17. Caution: For working with 32P, a ß-emitter, precautions should be taken to protect the environment and the technician. Wear protective glasses, use protective plexiglass screens and tube holders, and monitor benches for putative contamination using a Geiger-Müller tube (see Subheading 2.). Discard as short-lived radioactive waste.

18. Prepare a Sephadex G50 suspension by adding 30 g Sephadex to 500 mL of water. Autoclave and store at 4°C. Prepare a column by applying a small dot of glass wool on the narrow part inside of a Pasteur glass pipet (5-mm diameter). Pipet the Sephadex suspension in the pipet until the Sephadex level is approx 5 mm below the top of the Pasteur pipet. Equilibrate the column with 1 mL of TE before adding the end-labeling reaction mixture. Do not leave the column standing dry at any moment but continually keep adding 200-|L TE fractions. Collect fractions of 200 ||L in different tubes for radioactivity measurements.

19. Hybridize and wash at exactly 55°C. Higher temperatures may lead to negative results, as the probe may be unable to bind and lower temperatures may increase nonspecific background hybridization.

20. Radiolabeled probes in hybridization buffer can be stored at -20°C for reuse. Probes can be used several times, but keep in mind that 32P has a half-life of 14.3 d.


1. International Agency for Research on Cancer (1997) IARC monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 70: Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus/Human Herpesvirus 8. WHO, Lyon, France.

2 Middeldorp, J. M., Brink, A. A. T. P., van den Brule, A. J. C., and Meijer, C. J. L. M. (2003) Pathogenic roles for Epstein-Barr virus (EBV) gene products in EBV-associated proliferative disorders. Crit. Rev. Oncol. Hematol. 45, 1-36.

3 Oudejans, J. J., Jiwa, M., van den Brule, A. J., et al. (1995) Detection of heterogeneous Epstein-Barr virus gene expression patterns within individual post-transplantation lymphoproliferative disorders. Am. J. Pathol. 147, 923-933.

4 Brink, A. A., Oudejans, J. J., Jiwa, M., Walboomers, J. M., Meijer, C. J., and van den Brule, A. J. (1997) Multiprimed cDNA synthesis followed by PCR is the most suitable method for Epstein-Barr virus transcript analysis in small lymphoma biopsies. Mol. Cell. Probes 11, 39-47.

5 zur Hausen, A., Brink, A. A., Craanen, M. E., Middeldorp, J. M., Meijer, C. J., and van den Brule, A. J. (2000) Unique transcription pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric adenocarcinomas: expression of the transforming BARF1 gene. Cancer Res. 60, 2745-2748.

6 Brink, A. A. T. P., Vervoort, M. B. H. J., Middeldorp, J. M., Meijer, C. J. L. M., and van den Brule, A. J. C. (1998) Nucleic acid sequence based amplification (NASBA), a new method for analysis of spliced and unspliced Epstein-Barr virus latent transcripts and its comparison with RT-PCR. J. Clin. Microbiol. 36, 3164-3169.

7 Hayes, D. P., Brink, A. A. T. P., Vervoort, M. B. H. J., Middeldorp, J. M., Meijer, C. J. L. M., and van den Brule, A. J. C. (1999) Expression of Epstein-Barr virus (EBV) transcripts encoding homologues to important human proteins in diverse EBV-associated disease. Mol. Pathol. 52, 97-103.

8. Stevens, S. J. C, Blank, B. S., Smits, P. H., Meenhorst, P. L., and Middeldorp, J. M. (2002) High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 16, 993-1001.

9 Stevens, S. J. C, Verschuuren, E. A., Pronk, I., et al. (2001) Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients. Blood 97,1165-1171.

10 Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, and P. M., van der Noordaa, J. (1990) Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495-503.

11. Witt, D. J. and Kemper, M. (1999) Techniques for the evaluation of nucleic acid amplification technology performance with specimens containing interfering substances: efficacy of boom methodology for extraction of HIV-1 RNA. J. Virol. Methods 79, 97-111.

12. Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237-238.

Quantitative Detection of Viral Gene Expression in Populations of Epstein-Barr Virus-Infected Cells In Vivo

Donna R. Hochberg and David A. Thorley-Lawson


The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this technique involves limiting dilution analysis, it is possible to define which viral transcription programs are being used at the single-cell level. This assay takes 3-4 d to complete and involves the following steps: (1) sample preparation and isolation of the cell population of interest; (2) DNA-PCR limiting dilution analysis to determine the frequency of infected cells within the cell population; (3) RNA isolation; (4) cDNA synthesis; (5) PCR; (6) visualization of PCR products by Southern blotting; and (7) calculations. As an example, we have used PBMCs from the blood of an acute infectious mononucleosis patient. However, this technique can be applied to other cell populations, such as B cells, and other patient groups, such as healthy long-term virus carriers and immunosuppressed organ transplant recipients.

Key Words: EBV; Epstein-Barr virus; RT-PCR; DNA-PCR; infectious mononucleosis (IM); quantitative PCR; EBNA1; EBNA2; LMP1; LMP2; BZLF1; EBERs.

1. Introduction

The Epstein-Barr virus (EBV) is a ubiquitous, persistent virus implicated in a number of neoplasias including Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma (1). EBV is also the causative agent of infectious mononucleosis (IM) (2,3). In an effort to understand the etiology of these diseases, EBV has been the focus of much study both in clinically affected patients and in healthy long-term virus carriers. Early studies on tumor cells revealed that different transcription programs are expressed by the cells of different tumors (Table 1) (1). Virus-infected cells are relatively rare (4,5) in healthy carriers, making studies on this population more difficult. Continuing advances in polymerase chain reac-

From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ

Table 1

EBV Gene Expression Programs


Genes expressed

Found in:

Growth program (or latency III) Default program (or latency II) EBNA-1 only

Latency program (or Latency 0) Lytic program

All latent genes



All lytic genes

Cells infected in vitro Naive B cells of the tonsil Hodgkin's disease, NPC, germinal center and memory cells of the tonsil Burkitt's lymphoma, dividing memory

B cells of the peripheral blood Memory B cells of the peripheral blood

Plasma cells of the tonsil

Abbreviations: EBER, Epstein-Barr-encoded small RNA; EBNA, Epstein-Barr nuclear antigen; LMP, latent membrane protein; NPC, nasopharyngeal carcinoma.

tion (PCR) and reverse-transcription (RT)-PCR technologies have aided studies on the healthy population (5-7). By these approaches it is possible to detect mRNA and DNA from only a few copies of the target sequence or from a single infected cell. Such studies have shown that different populations of B cells in the healthy carrier express different transcription programs (Table 1) (8,9). These programs are the same as those originally described for different EBV-associated tumors. This information has allowed for the development of a comprehensive model of how EBV establishes and maintains a persistent infection while continuing to produce infectious virus. In this model EBV uses these transcription programs to mimic and induce normal B-cell differentiation in infected cells (10).

RT-PCR studies for EBV genes were and are generally performed on bulk cell populations (11,12). One shortfall of this approach is the inability to distinguish whether the amplification products are derived from transcripts present in a single cell or from many cells (13). This is because a typical sample will contain 106-107 B cells. Without knowing the number of infected cells present in the sample, it is impossible to determine whether a positive result means that all the infected cells are expressing a given gene or whether only a very small fraction of the infected cells are expressing the gene. Erroneous conclusions can be made from data collected in this way (see Note 30) (11-13). To determine the percentage of infected cells expressing a given gene, quantitative approaches are necessary. The first step in the quantitative RT-PCR method described below is to determine the number of infected cells present by limiting dilution DNA PCR for EBV. The next step is to perform limiting dilution RT-PCR for each gene. Poisson statistics are used to calculate the absolute number of EBV-infected cells and the percentage of these cells expressing the relevant viral

Table 2

Diagnostic Genes for EBV Expression Programsa


Gene expression program

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