Skip to main content
PMC home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1989 Dec;135(6):1035–1044.

Detection of Epstein-Barr virus by in situ hybridization. Progress toward development of a nonisotopic diagnostic test.

R Bashir 1, F Hochberg 1, R H Singer 1
PMCID: PMC1880496  PMID: 2556925

Abstract

This work presents some initial quantitation of an in situ hybridization method for detection of Epstein-Barr (EB) virus nucleic acids. The purpose is to develop evaluative criteria for diagnosis of viral presence in clinical tissue specimens. In this work simultaneous denaturation of probe and target DNA and an alkaline phosphatase conjugate to detect biotinated probe were used as described by Unger et al. For evaluation of the hybridization, a variety of cell lines, both productively and latently infected, that were hybridized in situ using nick translated 32P-labeled viral probe sequences and counted by scintillation after the method of Lawrence and Singer were used. Producer cells (B95-8) showed intense foci of staining in approximately 5% of cells, with most of the other cells showing varying staining intensity. Raji cells showed varying amounts of signal from cell to cell. Namalwa cells exhibited one spot in most cells that was decreased after cells were treated with Actinomycin D (dactinomycin, Merck Sharp & Dohme, West Point, PA). Signal was identified in only a third of these same cells after sectioning. EB virus-negative Ramos cells showed no signal. The nuclear punctate nature of the signal generated is diagnostic of infected cells, and may be a useful test for cultured cells or pathologic specimens.

Full text

PDF
1035

Images in this article

1040Figure 6
on p.1040
1042Figure 8
on p.1042

Selected References

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

  1. Andiman W., Gradoville L., Heston L., Neydorff R., Savage M. E., Kitchingman G., Shedd D., Miller G. Use of cloned probes to detect Epstein-Barr viral DNA in tissues of patients with neoplastic and lymphoproliferative diseases. J Infect Dis. 1983 Dec;148(6):967–977. doi: 10.1093/infdis/148.6.967. [DOI] [PubMed] [Google Scholar]
  2. Anvret M., Karlsson A., Bjursell G. Evidence for integrated EBV genomes in Raji cellular DNA. Nucleic Acids Res. 1984 Jan 25;12(2):1149–1161. doi: 10.1093/nar/12.2.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arrand J. R., Rymo L. Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells. J Virol. 1982 Feb;41(2):376–389. doi: 10.1128/jvi.41.2.376-389.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BURKITT D. A sarcoma involving the jaws in African children. Br J Surg. 1958 Nov;46(197):218–223. doi: 10.1002/bjs.18004619704. [DOI] [PubMed] [Google Scholar]
  5. BURKITT D. Determining the climatic limitations of a children's cancer common in Africa. Br Med J. 1962 Oct 20;2(5311):1019–1023. doi: 10.1136/bmj.2.5311.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  7. Bashir R. M., Harris N. L., Hochberg F. H., Singer R. M. Detection of Epstein-Barr virus in CNS lymphomas by in-situ hybridization. Neurology. 1989 Jun;39(6):813–817. doi: 10.1212/wnl.39.6.813. [DOI] [PubMed] [Google Scholar]
  8. Brahic M., Haase A. T. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6125–6129. doi: 10.1073/pnas.75.12.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. EPSTEIN M. A., ACHONG B. G., BARR Y. M. VIRUS PARTICLES IN CULTURED LYMPHOBLASTS FROM BURKITT'S LYMPHOMA. Lancet. 1964 Mar 28;1(7335):702–703. doi: 10.1016/s0140-6736(64)91524-7. [DOI] [PubMed] [Google Scholar]
  11. Epstein M. A., Hunt R. D., Rabin H. Pilot experiments with EB virus in owl monkeys (Aotus trivirgatus). I Reticuloproliferative disease in an inoculated animal. Int J Cancer. 1973 Sep 15;12(2):309–318. doi: 10.1002/ijc.2910120202. [DOI] [PubMed] [Google Scholar]
  12. Frank A., Andiman W. A., Miller G. Epstein-Barr virus and nonhuman primates: natural and experimental infection. Adv Cancer Res. 1976;23:171–201. doi: 10.1016/s0065-230x(08)60546-1. [DOI] [PubMed] [Google Scholar]
  13. Grogan E. A., Summers W. P., Dowling S., Shedd D., Gradoville L., Miller G. Two Epstein-Barr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7650–7653. doi: 10.1073/pnas.80.24.7650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanto D. W., Frizzera G., Purtilo D. T., Sakamoto K., Sullivan J. L., Saemundsen A. K., Klein G., Simmons R. L., Najarian J. S. Clinical spectrum of lymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein-Barr virus. Cancer Res. 1981 Nov;41(11 Pt 1):4253–4261. [PubMed] [Google Scholar]
  15. Henderson A., Ripley S., Heller M., Kieff E. Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1987–1991. doi: 10.1073/pnas.80.7.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Henle W., Henle G., Andersson J., Ernberg I., Klein G., Horwitz C. A., Marklund G., Rymo L., Wellinder C., Straus S. E. Antibody responses to Epstein-Barr virus-determined nuclear antigen (EBNA)-1 and EBNA-2 in acute and chronic Epstein-Barr virus infection. Proc Natl Acad Sci U S A. 1987 Jan;84(2):570–574. doi: 10.1073/pnas.84.2.570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hummel M., Kieff E. Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells. J Virol. 1982 Jul;43(1):262–272. doi: 10.1128/jvi.43.1.262-272.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. King W., Van Santen V., Kieff E. Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells. J Virol. 1981 May;38(2):649–660. doi: 10.1128/jvi.38.2.649-660.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klein G., Giovanella B., Westman A., Stehlin J. S., Mumford D. An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics. EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection. Intervirology. 1975;5(6):319–334. doi: 10.1159/000149930. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Lawrence J. B., Singer R. H. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 1985 Mar 11;13(5):1777–1799. doi: 10.1093/nar/13.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [DOI] [PubMed] [Google Scholar]
  24. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leibold W., Huldt G., Flanagan T. D., Andersson M., Dalens M., Wright D. H., Voller A., Klein G. Tumorigenicity of Epstein-Barr virus (EBV)-transformed lymphoid line cells in autologous squirrel monkeys. Int J Cancer. 1976 Apr 15;17(4):533–541. doi: 10.1002/ijc.2910170417. [DOI] [PubMed] [Google Scholar]
  26. Lindahl T., Adams A., Bjursell G., Bornkamm G. W., Kaschka-Dierich C., Jehn U. Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J Mol Biol. 1976 Apr 15;102(3):511–530. doi: 10.1016/0022-2836(76)90331-4. [DOI] [PubMed] [Google Scholar]
  27. Miller G., Shope T., Coope D., Waters L., Pagano J., Bornkamn G., Henle W. Lymphoma in cotton-top marmosets after inoculation with Epstein-Barr virus: tumor incidence, histologic spectrum antibody responses, demonstration of viral DNA, and characterization of viruses. J Exp Med. 1977 Apr 1;145(4):948–967. doi: 10.1084/jem.145.4.948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pritchett R., Pendersen M., Kieff E. Complexity of EBV homologous DNA in continous lymphoblastoid cell lines. Virology. 1976 Oct 1;74(1):227–231. [PubMed] [Google Scholar]
  29. Reedman B. M., Klein G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer. 1973 May;11(3):499–520. doi: 10.1002/ijc.2910110302. [DOI] [PubMed] [Google Scholar]
  30. Rosén A., Gergely P., Jondal M., Klein G., Britton S. Polyclonal Ig production after Epstein-Barr virus infection of human lymphocytes in vitro. Nature. 1977 May 5;267(5606):52–54. doi: 10.1038/267052a0. [DOI] [PubMed] [Google Scholar]
  31. Rymo L. Identification of transcribed regions of Epstein-Barr virus DNA in Burkitt lymphoma-derived cells. J Virol. 1979 Oct;32(1):8–18. doi: 10.1128/jvi.32.1.8-18.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Saemundsen A. K., Purtilo D. T., Sakamoto K., Sullivan J. L., Synnerholm A. C., Hanto D., Simmons R., Anvret M., Collins R., Klein G. Documentation of Epstein-Barr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by Epstein-Barr virus complementary RNA/DNA and viral DNA/DNA hybridization. Cancer Res. 1981 Nov;41(11 Pt 1):4237–4242. [PubMed] [Google Scholar]
  33. Sixbey J. W., Nedrud J. G., Raab-Traub N., Hanes R. A., Pagano J. S. Epstein-Barr virus replication in oropharyngeal epithelial cells. N Engl J Med. 1984 May 10;310(19):1225–1230. doi: 10.1056/NEJM198405103101905. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Unger E. R., Budgeon L. R., Myerson D., Brigati D. J. Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J Surg Pathol. 1986 Jan;10(1):1–8. doi: 10.1097/00000478-198601000-00001. [DOI] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES