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. 1991 Jun;44(6):487–491. doi: 10.1136/jcp.44.6.487

Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridisation.

P J Coates 1, W P Mak 1, G Slavin 1, A J d'Ardenne 1
PMCID: PMC496831  PMID: 1648578

Abstract

A highly sensitive non-isotopic in situ hybridisation technique was developed for the localisation of Epstein-Barr virus (EBV) in paraffin wax embedded tissue sections. The method uses a repeated sequence of the EBV genome as a probe, labelled with the novel reporter molecule, digoxigenin. The method can identify individual copies of EBV by detection of both EBV DNA and highly localised RNA transcripts. A combination of careful proteolytic digestion of tissue sections, high temperature denaturation of probe and target DNA, and sensitive immunocytochemical detection are used to attain single copy sensitivity. The technique is quicker and simpler to perform than some other methods used for the identification of EBV, and provides simultaneous morphological information which cannot be obtained by methods using tissue extracts. This method permits the investigation of the role of EBV in neoplastic conditions of lymphoid and epithelial cells, and may prove valuable in determining the sites of latent virus in healthy subjects.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allan G. M., Todd D., Smyth J. A., Mackie D. P., Burns J., McNulty M. S. In situ hybridization: an optimised detection protocol for a biotinylated DNA probe renders it more sensitive than a comparable 35S-labelled probe. J Virol Methods. 1989 Apr-May;24(1-2):181–190. doi: 10.1016/0166-0934(89)90020-7. [DOI] [PubMed] [Google Scholar]
  2. Anagnostopoulos I., Herbst H., Niedobitek G., Stein H. Demonstration of monoclonal EBV genomes in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization. Blood. 1989 Aug 1;74(2):810–816. [PubMed] [Google Scholar]
  3. Bashir R., Hochberg F., Singer R. H. Detection of Epstein-Barr virus by in situ hybridization. Progress toward development of a nonisotopic diagnostic test. Am J Pathol. 1989 Dec;135(6):1035–1044. [PMC free article] [PubMed] [Google Scholar]
  4. Brigati D. J., Myerson D., Leary J. J., Spalholz B., Travis S. Z., Fong C. K., Hsiung G. D., Ward D. C. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology. 1983 Apr 15;126(1):32–50. doi: 10.1016/0042-6822(83)90460-9. [DOI] [PubMed] [Google Scholar]
  5. Burns J., Graham A. K., Frank C., Fleming K. A., Evans M. F., McGee J. O. Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation. J Clin Pathol. 1987 Aug;40(8):858–864. doi: 10.1136/jcp.40.8.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheung A., Kieff E. Long internal direct repeat in Epstein-Barr virus DNA. J Virol. 1982 Oct;44(1):286–294. doi: 10.1128/jvi.44.1.286-294.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coates P. J., Hall P. A., Butler M. G., D'Ardenne A. J. Rapid technique of DNA-DNA in situ hybridisation on formalin fixed tissue sections using microwave irradiation. J Clin Pathol. 1987 Aug;40(8):865–869. doi: 10.1136/jcp.40.8.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coates P. J., d'Ardenne A. J., Khan G., Kangro H. O., Slavin G. Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections. J Clin Pathol. 1991 Feb;44(2):115–118. doi: 10.1136/jcp.44.2.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crum C. P., Nagai N., Levine R. U., Silverstein S. In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia. Am J Pathol. 1986 Apr;123(1):174–182. [PMC free article] [PubMed] [Google Scholar]
  10. Desgranges C., Wolf H., De-Thé G., Shanmugaratnam K., Cammoun N., Ellouz R., Klein G., Lennert K., Muñoz N., Zur Hausen H. Nasopharyngeal carcinoma. X. Presence of epstein-barr genomes in separated epithelial cells of tumours in patients from Singapore, Tunisia and Kenya. Int J Cancer. 1975 Jul 15;16(1):7–15. doi: 10.1002/ijc.2910160103. [DOI] [PubMed] [Google Scholar]
  11. Eversole L. R., Stone C. E., Beckman A. M. Detection of EBV and HPV DNA sequences in oral "hairy" leukoplakia by in situ hybridization. J Med Virol. 1988 Nov;26(3):271–277. doi: 10.1002/jmv.1890260307. [DOI] [PubMed] [Google Scholar]
  12. Furuta Y., Shinohara T., Sano K., Meguro M., Nagashima K. In situ hybridisation with digoxigenin-labelled DNA probes for detection of viral genomes. J Clin Pathol. 1990 Oct;43(10):806–809. doi: 10.1136/jcp.43.10.806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hamilton-Dutoit S. J., Pallesen G., Karkov J., Skinhøj P., Franzmann M. B., Pedersen C. Identification of EBV-DNA in tumour cells of AIDS-related lymphomas by in-situ hybridisation. Lancet. 1989 Mar 11;1(8637):554–552. doi: 10.1016/s0140-6736(89)90093-7. [DOI] [PubMed] [Google Scholar]
  14. Heiles H. B., Genersch E., Kessler C., Neumann R., Eggers H. J. In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. Biotechniques. 1988 Nov-Dec;6(10):978–981. [PubMed] [Google Scholar]
  15. Henle G., Henle W., Diehl V. Relation of Burkitt's tumor-associated herpes-ytpe virus to infectious mononucleosis. Proc Natl Acad Sci U S A. 1968 Jan;59(1):94–101. doi: 10.1073/pnas.59.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones J. F., Shurin S., Abramowsky C., Tubbs R. R., Sciotto C. G., Wahl R., Sands J., Gottman D., Katz B. Z., Sklar J. T-cell lymphomas containing Epstein-Barr viral DNA in patients with chronic Epstein-Barr virus infections. N Engl J Med. 1988 Mar 24;318(12):733–741. doi: 10.1056/NEJM198803243181203. [DOI] [PubMed] [Google Scholar]
  17. Knowles D. M., Inghirami G., Ubriaco A., Dalla-Favera R. Molecular genetic analysis of three AIDS-associated neoplasms of uncertain lineage demonstrates their B-cell derivation and the possible pathogenetic role of the Epstein-Barr virus. Blood. 1989 Feb 15;73(3):792–799. [PubMed] [Google Scholar]
  18. Krueger G. R., Papadakis T., Schaefer H. J. Persistent active Epstein-Barr virus infection and atypical lymphoproliferation. Report of two cases. Am J Surg Pathol. 1987 Dec;11(12):972–981. doi: 10.1097/00000478-198712000-00008. [DOI] [PubMed] [Google Scholar]
  19. Lawrence J. B., Singer R. H., Marselle L. M. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell. 1989 May 5;57(3):493–502. doi: 10.1016/0092-8674(89)90924-0. [DOI] [PubMed] [Google Scholar]
  20. Montone K. T., Budgeon L. R., Brigati D. J. Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions. Mod Pathol. 1990 Jan;3(1):89–96. [PubMed] [Google Scholar]
  21. Morris R. G., Arends M. J., Bishop P. E., Sizer K., Duvall E., Bird C. C. Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation. J Clin Pathol. 1990 Oct;43(10):800–805. doi: 10.1136/jcp.43.10.800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mueller N., Evans A., Harris N. L., Comstock G. W., Jellum E., Magnus K., Orentreich N., Polk B. F., Vogelman J. Hodgkin's disease and Epstein-Barr virus. Altered antibody pattern before diagnosis. N Engl J Med. 1989 Mar 16;320(11):689–695. doi: 10.1056/NEJM198903163201103. [DOI] [PubMed] [Google Scholar]
  23. Murphy J. K., Young L. S., Bevan I. S., Lewis F. A., Dockey D., Ironside J. W., O'Brien C. J., Wells M. Demonstration of Epstein-Barr virus in primary brain lymphoma by in situ DNA hybridisation in paraffin wax embedded tissue. J Clin Pathol. 1990 Mar;43(3):220–223. doi: 10.1136/jcp.43.3.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Niedobitek G., Hamilton-Dutoit S., Herbst H., Finn T., Vetner M., Pallesen G., Stein H. Identification of Epstein-Barr virus-infected cells in tonsils of acute infectious mononucleosis by in situ hybridization. Hum Pathol. 1989 Aug;20(8):796–799. doi: 10.1016/0046-8177(89)90075-0. [DOI] [PubMed] [Google Scholar]
  25. Patton D. F., Wilkowski C. W., Hanson C. A., Shapiro R., Gajl-Peczalska K. J., Filipovich A. H., McClain K. L. Epstein-Barr virus--determined clonality in posttransplant lymphoproliferative disease. Transplantation. 1990 Jun;49(6):1080–1084. doi: 10.1097/00007890-199006000-00010. [DOI] [PubMed] [Google Scholar]
  26. Peiper S. C., Myers J. L., Broussard E. E., Sixbey J. W. Detection of Epstein-Barr virus genomes in archival tissues by polymerase chain reaction. Arch Pathol Lab Med. 1990 Jul;114(7):711–714. [PubMed] [Google Scholar]
  27. Rogers B. B., Alpert L. C., Hine E. A., Buffone G. J. Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am J Pathol. 1990 Mar;136(3):541–548. [PMC free article] [PubMed] [Google Scholar]
  28. Teo C. G., Griffin B. E. Epstein-Barr virus genomes in lymphoid cells: activation in mitosis and chromosomal location. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8473–8477. doi: 10.1073/pnas.84.23.8473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thorley-Lawson D. A. Immunological responses to Epstein-Barr virus infection and the pathogenesis of EBV-induced diseases. Biochim Biophys Acta. 1989 Feb;948(3):263–286. doi: 10.1016/0304-419x(89)90002-4. [DOI] [PubMed] [Google Scholar]
  30. Venables P. J., Teo C. G., Baboonian C., Griffin B. E., Hughes R. A. Persistence of Epstein-Barr virus in salivary gland biopsies from healthy individuals and patients with Sjögren's syndrome. Clin Exp Immunol. 1989 Mar;75(3):359–364. [PMC free article] [PubMed] [Google Scholar]
  31. Walboomers J. M., Melchers W. J., Mullink H., Meijer C. J., Struyk A., Quint W. G., van der Noordaa J., ter Schegget J. Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squamous cell carcinomas of the cervix. Am J Pathol. 1988 Jun;131(3):587–594. [PMC free article] [PubMed] [Google Scholar]
  32. Warford A. In situ hybridisation: a new tool in pathology. Med Lab Sci. 1988 Oct;45(4):381–394. [PubMed] [Google Scholar]
  33. Weiss L. M., Movahed L. A., Butler A. E., Swanson S. A., Frierson H. F., Jr, Cooper P. H., Colby T. V., Mills S. E. Analysis of lymphoepithelioma and lymphoepithelioma-like carcinomas for Epstein-Barr viral genomes by in situ hybridization. Am J Surg Pathol. 1989 Aug;13(8):625–631. doi: 10.1097/00000478-198908000-00001. [DOI] [PubMed] [Google Scholar]
  34. Weiss L. M., Movahed L. A. In situ demonstration of Epstein-Barr viral genomes in viral-associated B cell lymphoproliferations. Am J Pathol. 1989 Mar;134(3):651–659. [PMC free article] [PubMed] [Google Scholar]
  35. Weiss L. M., Movahed L. A., Warnke R. A., Sklar J. Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med. 1989 Feb 23;320(8):502–506. doi: 10.1056/NEJM198902233200806. [DOI] [PubMed] [Google Scholar]
  36. Weiss L. M., Strickler J. G., Warnke R. A., Purtilo D. T., Sklar J. Epstein-Barr viral DNA in tissues of Hodgkin's disease. Am J Pathol. 1987 Oct;129(1):86–91. [PMC free article] [PubMed] [Google Scholar]

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