141

Susanne M. Bailer and Diana Lieber (eds.), Virus-Host Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 1064, DOI 10.1007/978-1-62703-601-6_10, © Springer Science+Business Media, LLC 2013

Chapter 10

Detection of Integrated Herpesvirus Genomes by Fluorescence In Situ Hybridization (FISH)

Benedikt B. Kaufer

Abstract

Fluorescence in situ hybridization (FISH) is widely used to visualize nucleotide sequences in interphase cells or on metaphase chromosomes using specifi c probes that are complementary to the respective targets. Besides its broad application in cytogenetics and cancer research, FISH facilitates the localization of virus genomes in infected cells. Some herpesviruses, including human herpesvirus 6 (HHV-6) and Marek’s disease virus (MDV), have been shown to integrate their genetic material into host chromosomes, which allows transmission of HHV-6 via the germ line and is required for effi cient MDV-induced tumor formation. We describe here the detection by FISH of integrated herpesvirus genomes in metaphase chromosomes and interphase nuclei of herpesvirus-infected cells.

Key words Fluorescence in situ hybridization , Metaphase spread , Herpesvirus , Integration , Telomere , Telomeric repeats , Marek’s disease virus , Human herpesvirus 6 , Lymphoblastoid cell lines

1 Introduction

Fluorescence in situ hybridization (FISH) facilitates detection of specifi c DNA and RNA sequences in interphase cells or meta-phase chromosomes. It is a widely used tool in various research areas including cancer research, pre- and postnatal diagnostics, cytogenetics, developmental biology, gene mapping and virology [ 1 , 2 ]. In virology, FISH allows detection of viral genomic sequences in infected cells during various stages of the virus life cycle [ 3 – 7 ].

Several DNA viruses and retroviruses have been shown to inte-grate their genetic material into host chromosomes, which is required for effi cient replication and maintenance of the viral genome in proliferating cells [ 8 , 9 ]. Some herpesviruses, including human her-pesvirus 6 (HHV-6) and the lymphoma-inducing Marek’s disease virus (MDV), have been shown to integrate their genetic material into host chromosomes [ 10 – 12 ]. Integration of these two herpesvi-ruses has recently been shown to occur in host telomeres, a

142

protective structure at the end of linear chromosomes [ 13 , 14 ]. In case of HHV-6, integrated virus genomes are present in the germ line of about 1 % of the human population, a consequence and prerequisite for vertical transmission of the virus from parents to offspring [ 8 , 14 ]. Intriguingly, HHV-6 and MDV genome inte-gration is not a dead end as both viruses can reactivate from cells that contain only integrated virus genomes, resulting in lytic virus replication [ 14 , 15 ]; however, the exact mechanism of and cues for genome mobilization from the integrated state are unknown.

Integrated herpesvirus genomes can be readily detected in metaphase chromosomes of MDV transformed cells by FISH (Fig. 1a, b ), while no signal is detected in chromosomes of cells that do not harbor the MDV genome (Fig. 1c ). In addition, FISH can be used to visualize viral DNA in lytically infected cells and upon reactivation of the virus from the latent stage of infection. During lytic replication, the virus genome is massively amplifi ed and can be detected as a strong fl uorescence signal all over interphase nuclei, while only defi ned spots can be detected in the

Fig. 1 Fluorescence in situ hybridization analysis detecting MDV integration sites (anti-DIG FITC, green ) on metaphase chromosomes (DAPI stain, blue ) performed on MDV-induced or control lymphoblastoid cell lines (LCLs). ( a and b ) MDV-induced LCLs harboring several ( a ) or only a single ( b ) MDV integration site. ( c ) Metaphase chromosomes of negative control cell line CU91. ( d and e ) MDV reactivation resulting in large amounts of viral genomic DNA detectable in some but not all interphase nuclei. ( f ) Interphase nucleus of CU91 negative control cell line

Benedikt B. Kaufer

143

Table 1 Troubleshooting guide

Observation/problem Potential cause Suggestion

No or very weak staining

Not enough probe Increase amount of probe during hybridization

Insuffi cient probe labeling Test the probe by dot blot hybridization Incomplete chromosome or

probe denaturation Control denaturation temperature and

denaturation buffer composition Stringency of post- hybridization

washes too high Decrease temperature of stringency

washes Increase salt concentration of SSC wash

buffer (e.g., 2–4× SSC) Decrease duration of the wash steps

Background staining

1. On chromosomes or nuclei

Low stringency of post-hybridization washes

Decrease salt concentration of SSC wash buffer (e.g., 0.5–1.0× SSC)

Increase stringency of wash temperature Increase duration of the wash steps

Insuffi cient blocking Increase amount of salmon sperm or Cot-1 DNA

Hybridization conditions Increase hybridization temperature Decrease hybridization time

Too much probe Decrease probe concentration

2. Around the chromosomes and the nuclei, but not on the slide

Suboptimal cell preparation with a lot of cell debris

Increase hypotonic treatment Increase pepsin treatment

DNA probe fragments are too long

Generate shorter probes

3. Generalized background on slides but not at chromosomes

Insuffi cient post- hybridization wash

Change wash buffer at least 3 times Shake coplin jar to ensure proper

washing Antibody concentration too

high Reduce antibody concentration

4. Spotty background over the slide

Excessive slide aging Less stringent aging conditions Try chemical aging with ethanol

Probe gets trapped in protein aggregate

Increase pepsin treatment

Antibody aggregates Centrifuge antibody stock solutions to remove aggregates

nuclei of latently infected cells (Fig. 1d, e ). No signal is detected in interphase nuclei that do not harbor the MDV genome (Fig. 1f ).

This chapter describes the detection of herpesvirus genomes by FISH, including a detailed description of the preparation of metaphase spreads, generation of herpesvirus-specifi c probes, denaturation and hybridization of DNA probes to herpesvirus genomes, and the analysis of the samples via fl uorescence micros-copy. Potential problems are discussed in Subheading 4 and the troubleshooting guide (Table 1 ).

FISH of Herpesvirus Integration

144

2 Materials

1. LM-Hahn medium: Combine 500 mL McCoy’s 5A medium, 500 mL Leibovitz L-15, 100 mL 10× tryptose phosphate broth, 2.6 mL 10 % Na-bicarbonate, 11 mL 1 mM β-mercaptoethanol, 11 mL 100× NaPyruvate (11 g/L H 2 O), 11 mL Antibiotics-Antimycotics solution, 11 mL 200 mM L -Glutamine, 110 mL fetal bovine serum (FBS) and 88 mL chicken serum [ 16 ].

2. RPMI 1640 medium supplemented with 10 % FBS and 100 U/mL penicillin and 0.1 mg/mL streptomycin.

3. Ultrapure deionized water (ddH 2 O; see Note 1 ). 4. Phosphate buffered saline (PBS). Dissolve 8.0 g NaCl, 0.2 g

KCl, 1.44 g Na 2 HPO 4 and 0.24 g KH 2 PO 4 in 800 mL ddH 2 O. Adjust the pH to 7.4 with 1 M HCl and bring to a fi nal volume of 1 L with ddH 2 O. Prepare aliquots, autoclave and store at room temperature.

5. Colcemid solution (10 µg/mL colcemid in PBS). 6. Hypotonic solution: 0.075 M KCl in ddH 2 O. 7. Fixative: Methanol:acetic acid (3:1, v/v), store at −20 °C. 8. Precleaned microscope slides (e.g., Superfrost, Fisher Scientifi c). 9. Adjustable water bath (range: 20–80 °C). 10. Phase contrast microscope.

1. Purifi ed herpesvirus BAC, cosmid or viral DNA ( see Note 2 ). 2. Frequently cutting restriction enzymes (4-bp recognition sites),

e.g., HaeIII or DpnI . 3. DNA purifi cation kit (e.g., QIAquick PCR Purifi cation Kit,

Qiagen). 4. Biotin or DIG-High Prime random priming labeling kit (Roche).

1. 22 × 22 mm glass coverslips. 2. Rubber cement. 3. Coplin jars (50 mL) optimally with a screw cap. 4. Glass bowl. 5. Humidifi ed chamber (airtight container containing a damp

paper towel or blotting paper). 6. Incubators at 37 and 80 °C. 7. Ethanol series: 70, 90 and 100 % ethanol in ddH 2 O. 8. Pepsin solution: 0.005 % pepsin in 10 mM HCl. 9. 20× SSC buffer: Dissolve 175.3 g NaCl and 88.2 g Na-citrate

in 800 mL ddH 2 O. Adjust the pH to 7.0 with 1 M HCl and

2.1 Metaphase Chromosome Preparation

2.2 FISH Probes

2.3 Fluorescence In Situ Hybridization

Benedikt B. Kaufer

145

bring to a fi nal volume of 1 L with ddH 2 O. Prepare aliquots, autoclave and store at room temperature.

10. 2× SSC wash buffer: Mix 100 mL of 20× SSC and 900 mL ddH 2 O and store at room temperature.

11. 0.5× SSC wash buffer: Mix 25 mL of 20× SSC and 975 mL ddH 2 O and store at room temperature.

12. Denaturation buffer: Mix 70 mL deionized formamide and 30 mL of 2× SSC and store at 4 °C ( see Note 3 ).

13. Phosphate buffer: Mix 80 mL 500 mM sodium phosphate dibasic solution with 20 mL sodium phosphate monobasic solution to obtain pH 7.0. Prepare aliquots, autoclave and store at room temperature.

14. Dextran sulfate stock solution (50 %): Dissolve 25 g dextran sulfate in 40 mL ddH 2 O by stirring over night at 4 °C. Adjust volume to 50 mL with ddH 2 O, aliquot and store at −20 °C.

15. Hybridization buffer: Mix 50 mL deionized formamide, 10 mL 20× SSC, 10 mL phosphate buffer pH 7.0, 16 mL dextran sul-fate stock solution (50 %) and bring to a fi nal volume of 100 mL with ddH 2 O. Aliquot the solution and store at −20 °C.

16. Sheared salmon sperm or Cot-1 DNA (20 mg/mL). 17. Stringency wash buffer: Mix 50 mL deionized formamide and

50 mL 2× SSC and store at 4 °C. 18. Detergent wash buffer: Mix 1 L of 4× SSC and 500 µL

Tween- 20 and store at room temperature. 19. Antibody solution: Dilute fl uorescently labeled avidin or anti-

digoxigenin antibody in detergent wash buffer 1:500 or according to the manufacturer’s instruction (Sigma-Aldrich; see Note 4 ).

20. Mounting solution containing DAPI 4,6-diamidino-2- phenylindole (e.g., DAPI Vectashield, Vector).

21. Fluorescence microscope containing appropriate fi lter sets for all fl uorophores used, a 60× or 100× oil immersion objective and a CCD (charge coupled device) camera.

3 Methods

1. Propagate MDV or HHV-6 infected cells or lymphoblastoid cell lines (LCLs) in the appropriate medium and passage cells 1 day prior to metaphase preparation. Alternatively, cells can be directly isolated ex vivo ( see Note 5 ).

2. Add 0.1 µg/mL colcemid to the cell suspension and incubate for 1–3 h for enrichment of metaphase cells ( see Note 6 ).

3. Transfer 1 × 10 6 to 1 × 10 7 suspension cells into a 15 mL conical tube and centrifuge at 400 × g for 8 min at room temperature.

3.1 Preparation of Cells

FISH of Herpesvirus Integration

146

4. Discard the supernatant and fl ick the tube to loosen the cell pellet.

5. Carefully resuspend cells in 10 mL prewarmed hypotonic solution and incubate for 10–15 min at 37 °C ( see Note 7 ).

6. Add 1 mL fresh ice-cold fi xative solution and mix by gently inverting the tube to fi x the cells and prevent cell clumping.

7. Centrifuge at 400 × g for 8 min at room temperature. Discard supernatant and fl ick the tube to loosen the cell pellet.

8. Carefully add 5 mL fresh ice-cold fi xative solution along the wall of the tube.

9. Centrifuge at 400 × g for 8 min at room temperature. Discard the supernatant and fl ick the tube to loosen the cell pellet.

10. Repeat steps 8 and 9 two more times. 11. Resuspend cells in 1–2 mL ice-cold fi xative solution ( see Note 8 ). 12. Samples can be stored at −20 °C for several years ( see Note 9 ).

1. Prewarm water bath to 80 °C. 2. Resuspend metaphase cell preparation by flicking the tube

( see Note 10 ). 3. Drop 10–20 µL of cell suspension onto a precleaned micro-

scope slide. 4. After the slide surface becomes “grainy”, move the slide briefl y

through the vapor of the water bath for 1–2 s and dry the slides at room temperature ( see Note 11 ).

5. Check the quality of the metaphase spreads using a phase- contrast microscope. Proceed with the best preparations or adjust conditions to improve slide quality ( see Note 12 ).

6. Dehydrate slide in 100 % ethanol for 5 min. 7. Dry at room temperature. 8. Incubate slide in pepsin solution for 2–5 min ( see Note 13 ). 9. Wash slide three times in fresh 2× SSC buffer for 1 min each. 10. Briefl y immerse slide in ddH 2 O. 11. Dehydrate slides twice in 70 % ethanol for 2 min each, then

twice in 90 % ethanol for 2 min each, and fi nally in 100 % ethanol for 4 min at room temperature.

12. Dry the slide at room temperature. 13. Age the slide at 65 °C on a hot plate for 1 h ( see Note 14 ).

1. Digest 2 µg of purifi ed herpesvirus BAC, cosmid or viral DNA with the appropriate restriction enzyme (e.g., HaeIII or DpnI ) for 2 h ( see Note 15 ).

2. Purify digested DNA with a DNA purifi cation kit.

3.2 Preparation of Metaphase Chromosome Spread Slides

3.3 Preparation of FISH Probes (Random Priming)

Benedikt B. Kaufer

147

3. Analyze aliquot of digested DNA on a 1 % agarose gel ( see Note 16 ).

4. Dilute 1–2 µg of the DNA in 16 µL ddH 2 O and denature for 10 min at 95 °C. Immediately cool the denatured DNA in an ice bath.

5. Mix DNA solution with 4 µL Biotin or DIG-High Prime and incubate over night at 37 °C ( see Note 17 ).

6. Purify digested DNA with a DNA purifi cation kit to remove unincorporated nucleotides ( see Note 18 ).

1. Prepare staining solution by mixing 20–40 ng of DIG- labeled probe and 20 µg of sheared salmon sperm or Cot-1 DNA in 12 µL hybridization buffer ( see Note 19 ).

2. Denature staining solution for 5 min at 75 °C and immediately cool the DNA in an ice bath.

3. Add 12 µL of the staining solution onto a coverslip and lower it onto the metaphase slide ( see Note 20 ).

4. Seal coverslip with rubber cement and let dry at room tempera-ture ( see Note 21 ).

5. Place slides on a metal surface in the 80 °C incubator and incu-bate for 2 min ( see Note 22 ).

6. Open the incubator door slightly (10 mm) and let it cool to 37 °C over approximately 15 min.

7. Transfer slides into a humidity chamber in a preheated 37 °C incubator and incubate for 24–48 h ( see Note 23 ).

1. Prewarm two coplin jars containing 50 mL stringency wash buffer and two coplin jars with 2× SSC wash buffer in a 42 °C water bath.

2. Remove the slides from the humidity chamber and place them into a glass bowl containing 2× SSC wash buffer. Carefully remove the rubber cement and coverslip ( see Note 24 ).

3. Transfer slides into prewarmed coplin jar containing the strin-gency wash buffer and incubate for 5 min at 42 °C. Transfer slides into the second stringency wash buffer jar and repeat wash step ( see Note 25 ).

4. Wash slides twice for 5 min each in prewarmed 2× SSC wash buffer.

5. Incubate slides for 5 min in detergent wash buffer. If directly labeled probes were used, proceed to step 8 .

6. Drain slides, apply 75 µL of antibody solution and cover imme-diately with a piece of parafi lm. Incubate the slide in the humidity chamber for 15–20 min at 37 °C ( see Note 26 ).

3.4 Hybridization

3.5 Wash and Detection

FISH of Herpesvirus Integration

148

7. Remove parafi lm from the slide and wash three times with detergent wash buffer in a coplin jar for 4 min each at room temperature.

8. Briefl y immerse slide in ddH 2 O and let dry at room temperature.

9. Mount slide with 25 µL Vectashield DAPI using a coverslip. 10. Examine FISH slides using a fl uorescence microscope, record

images and determine the number and locations of the herpes-virus integration sites. Individual chromosomes can be distin-guished by their size or by labeling each chromosome with a specifi c probe. Reactivation of herpesvirus genomes can be readily detected in interphase nuclei (Fig. 1e, f ). If no or very weak signal is detectable or if background is present on the slide, follow the troubleshooting guide in Table 1 .

4 Notes

1. All buffers and reagents should be prepared using ultrapure ddH 2 O with a resistance of 18.2 MΩ cm at 25 °C.

2. The purity of the viral DNA is crucial for the generation of FISH probes. Contamination with cellular DNA can cause unspecifi c background staining. Optimally, BACs or cosmid clones contain-ing the herpesvirus genome should be used as a template for the generation of FISH probes. DNA of BAC or cosmid clones should be purifi ed using silica column-based DNA extraction kits to ensure the purity and quality of the DNA.

3. Use high-quality deionized formamide for the preparation of the denaturation buffer to ensure an effi cient denaturation of the target DNA. Low quality formamide can result in the dis-tortion or thickening of the metaphase chromosomes [ 17 ]. Handle solutions containing formamide only in the fume hood as it can cause respiratory tract, eye and skin irritation.

4. Fluorescently labeled avidin and anti-digoxigenin antibodies are commercially (e.g., Sigma-Aldrich, Roche Applied Science) available with a variety of fl uorophores including FITC, Cy3 and Cy5. Antibody precipitates can result in unspecifi c dotted background on FISH slide and should be removed by centrifu-gation of the antibody solution for 5 min at >10,000 × g prior to use.

5. Propagate HHV-6 infected cells and LCLs in RPMI medium containing 10 % FBS at 37 °C under a 5 % CO 2 atmosphere. MDV infected LCLs should be propagated in LM Hahn media at 41 °C. HHV-6 and MDV infected lymphocytes can be stimulated using 0.5–5 µg/mL phytohemagglutinin (PHA) over night to increase the number of cells in metaphase.

Benedikt B. Kaufer

149

As shown in Fig. 1d, e , the virus genome can also be detected in the nucleus of interphase cells; however, it is impossible to determine if it is associated with cellular chromosomes.

6. If only few metaphase spreads are detectable, extend colcemid treatment to 12 or 24 h in order to increase the number of cells in metaphase. However, prolonged treatment can lead to chromosome breaks [ 18 ] resulting in very short chromosomes in metaphase spread preparations. If adherent cells are being used, trypsinize the cells, wash them two times with PBS, and proceed with the next step.

7. Duration and temperature of the hypotonic treatment have a strong effect on the quality of metaphase preparations. Insuffi cient hypotonic treatment results in metaphase chromo-somes that do not spread well and are surrounded by a large amount of cytoplasmic debris. The proteinaceous debris can prevent the FISH probe from gaining access to target sequences. If the hypotonic treatment is too long, chromo-somes may appear thicker and swollen. Adjust hypotonic treat-ment accordingly.

8. If very few metaphase chromosomes and interphase nuclei are present upon spreading on microscope slides, centrifuge the cells and resuspend in a smaller volume of fi xative solution. Add more fi xative solution if the cell suspension is too dense.

9. If samples were stored for more than 2 weeks, centrifuge cells at 400 × g , discard supernatant and resuspend cells in fresh fi xa-tive solution. This ensures the optimal methanol:acetic acid ratio required for high-quality metaphase spreads. Cells can be transferred into 2 mL tubes for long-term storage at −20 °C.

10. Ensure that cell clumps are completely resuspended. Cell clumping can drastically reduce the number of metaphase spreads on the slides.

11. Evaporation of the fi xative solution facilitates spreading of the metaphase chromosomes on the microscope slides. Humidity plays a crucial role during this process, which can be optimally controlled using the 80 °C water bath. The drying temperature can be modifi ed to optimize spread of metaphase chromosomes as described previously [ 17 ].

12. Metaphase chromosomes should be clearly visible on the micro-scope slide using a phase-contrast microscope. If metaphase chro-mosomes do not spread well and remain as a clump on the slide, you may either increase hypotonic treatment ( see Note 7 ), modify the length of vapor application or drying temperature for the slides ( see Subheading 3.2 , step 4 and Note 11 ). If cytoplasmic debris is visible as a halo covering the metaphase chromosomes, increase the time of the hypotonic treatment to 15–20 min ( see Note 7 ) or extend pepsin treatment ( see Note 13 ).

FISH of Herpesvirus Integration

150

13. Pepsin treatment allows removal of protein debris covering the chromosomes that can prevent the FISH probe from gaining access to target sequences. Pepsin treatment of metaphase spread slides with a large amount of cytoplasmic debris should be extended to 5–20 min. Excessive pepsin treatment can result in loss of chromosomes or chromosomal structure.

14. Aging of the slides is another crucial step that facilitates fi xation of the chromosomes and interphase nuclei onto the micro-scope slide. It is also important for the preservation of the chromosome architecture. Excessive aging enhances the rigidity of the chromosomes, which can decrease the hybridization effi ciency of the FISH probe to the chromosomes.

15. Large FISH probes usually bind less specifi cally and less effi -ciently to chromosomes than shorter probes. Digestion of purifi ed herpesvirus BAC, cosmid or viral DNA with frequent cutters ensures an optimal probe size of <2,000 bp.

16. Analyze the DNA by gel electrophoresis to ensure that the quality is suffi cient and that the DNA is completely digested.

17. Incubation can be extended to 24 h to increase the yield of the labeled probe. Alternatively, probes can be also directly labeled using commercially available nucleotides such as Fluorescein-12- dUTP, Cy3-6-dUTP or Cy5-dUTP. Signal intensities of directly labeled probes are usually lower compared to hapten- labeled probes, as every avidin- or hapten-specifi c antibody carries several fl uorophores. Various FISH labeling methods and fl uorophore selection have been reviewed by Morrison and colleagues [ 19 ].

18. Presence of unincorporated labeled nucleotides results in back-ground staining on FISH slides. Alternatively, unincorporated nucleotides can be removed using Sephadex G-50 columns or by ethanol precipitation [ 19 ].

19. Denaturation and hybridization can be performed separately as described previously [ 20 ]. This sequential staining procedure provides comparable results; however, it is more labor- intensive than the simultaneous staining described here.

20. Air bubbles trapped under the coverslip will result in uneven staining of the slide. If air bubbles are present, gently apply pressure to the coverslip with a pipette tip to remove the bubbles.

21. Samples should not dry during the staining procedure. Make sure that the coverslip is completely sealed with rubber cement to avoid drying of the slides in the following steps.

22. Complete denaturation is essential for effi cient hybridization of the FISH probe to the chromosomes. Measure the tempera-ture adjacent to the slides with a thermometer to ensure proper denaturation of the samples.

Benedikt B. Kaufer

151

23. Usage of a humidity chamber ensures that the slides do not dry out. Hybridization temperature can be increased if unspe-cifi c chromosomal staining is detectable. Control the tempera-ture of the incubator to ensure the optimal hybridization temperature.

24. The slides should be covered with 2× SSC wash buffer to ensure that they do not dry out. Remove the coverslips very carefully as the samples can be damaged during this step.

25. Stringency wash steps facilitate removal of unspecifi c-bound probes. Stringency should be increased if unspecifi c chromo-somal staining is observed by raising the stringency wash tem-perature up to 65 °C, reducing the salt concentration during the SSC wash steps ( see Subheading 3.3 , step 4 ) by using 0.5× or 1× SSC wash buffers, or by extending the duration of wash steps. Control the temperature of the wash solutions to ensure optimal wash conditions.

26. Ensure that the slides do not dry out during the staining pro-cedure as this would result in unspecifi c background staining on the slides.

Acknowledgments

The author thanks Dr. Nikolaus Osterrieder for editing the manu-script. This work was supported by the DFG grant KA3492.1-1 and funding from the Freie Universität Berlin to B.B.K.

References

1. Trask BJ (1991) Fluorescence in situ hybridiza-tion: applications in cytogenetics and gene mapping. Trends Genet 7(5):149–154

2. Wiegant J, Ried T, Nederlof PM et al (1991) In situ hybridization with fl uoresceinated DNA. Nucleic Acids Res 19(12):3237–3241

3. Hackstein H, Jahn G, Kirchner H et al (1996) Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalo-virus DNA in peripheral blood leukocytes. Histochem Cell Biol 106(2):229–234

4. Lawrence JB, Marselle LM, Byron KS et al (1990) Subcellular localization of low-abundance human immunodefi ciency virus nucleic acid sequences visualized by fl uores-cence in situ hybridization. Proc Natl Acad Sci USA 87(14):5420–5424

5. Reisinger J, Rumpler S, Lion T et al (2006) Visualization of episomal and integrated Epstein-Barr virus DNA by fi ber fl uorescence

in situ hybridization. Int J Cancer 118(7):1603–1608

6. Brabec-Zaruba M, Pfanzagl B, Blaas D et al (2009) Site of human rhinovirus RNA uncoat-ing revealed by fl uorescent in situ hybridization. J Virol 83(8):3770–3777

7. Robertson KL, Verhoeven AB, Thach DC et al (2010) Monitoring viral RNA in infected cells with LNA fl ow-FISH. RNA 16(8):1679–1685

8. Hall CB, Caserta MT, Schnabel K et al (2008) Chromosomal integration of human herpesvirus 6 is the major mode of congenital human her-pesvirus 6 infection. Pediatrics 122(3):513–520

9. Li M, Mizuuchi M, Burke TR Jr et al (2006) Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J 25(6):1295–1304

10. Delecluse HJ, Hammerschmidt W (1993) Status of Marek’s disease virus in established lymphoma cell lines: herpesvirus integration is common. J Virol 67(1):82–92

FISH of Herpesvirus Integration

152

11. Luppi M, Barozzi P, Marasca R et al (1994) Integration of human herpesvirus-6 (HHV-6) genome in chromosome 17 in two lymphoma patients. Leukemia 8(Suppl 1):S41–S45

12. Luppi M, Marasca R, Barozzi P et al (1993) Three cases of human herpesvirus-6 latent infection: integration of viral genome in periph-eral blood mononuclear cell DNA. J Med Virol 40(1):44–52

13. Kaufer BB, Jarosinski KW, Osterrieder N (2011) Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation. J Exp Med 208(3):605–615

14. Arbuckle JH, Medveczky MM, Luka J et al (2010) The latent human herpesvirus-6A genome specifi cally integrates in telomeres of human chromosomes in vivo and in vitro. Proc Natl Acad Sci USA 107(12):5563–5568

15. Delecluse HJ, Schuller S, Hammerschmidt W (1993) Latent Marek’s disease virus can be activated from its chromosomally integrated

state in herpesvirus-transformed lymphoma cells. EMBO J 12(8):3277–3286

16. Calnek BW, Shek WR, Schat KA (1981) Spontaneous and induced herpesvirus genome expression in Marek’s disease tumor cell lines. Infect Immun 34(2):483–491

17. Henegariu O, Heerema NA, Lowe WL et al (2001) Improvements in cytogenetic slide preparation: controlled chromosome spread-ing, chemical aging and gradual denaturing. Cytometry 43(2):101–109

18. Satya-Prakash KL, Liang JC, Hsu TC et al (1986) Chromosome aberrations in mouse bone marrow cells following treatment in vivo with vinblastine and Colcemid. Environ Mutagen 8(2):273–282

19. Morrison LE, Ramakrishnan R, Ruffalo TM et al (2002) Labeling fl uorescence in situ hybridization probes for genomic targets. Methods Mol Biol 204:21–40

20. Rens W, Fu B, O’Brien PC et al (2006) Cross- species chromosome painting. Nat Protoc 1(2):783–790

Benedikt B. Kaufer