Skip to main content
SpringerLink
Log in

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH

  • Original Article
  • Published:
International Journal of Hematology Aims and scope Submit manuscript

Abstract

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data availability

The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.

Abbreviations

CAEBV:

Chronic active EBV disease

EBERs:

EBV-encoded small RNAs

EBV:

Epstein-Barr virus

EBV-HLH:

EBV-associated hemophagocytic lymphohistiocytosis

HCT:

Hematopoietic cell transplantation

HV-LPD:

Hydroa vacciniforme lymphoproliferative disorder

IM:

Infectious mononucleosis

PTLD:

Posttransplant lymphoproliferative disorder

qPCR:

Quantitative PCR

SMBA:

Severe mosquito bite allergy

XLP1:

X-linked lymphoproliferative disease type 1

References

  1. Cohen JI. Epstein-Barr virus infection. N Engl J Med. 2000;343:481–92.

    Article  CAS  PubMed  Google Scholar 

  2. Fournier B, Latour S. Immunity to EBV as revealed by immunedeficiencies. Curr Opin Immunol. 2021;72:107–15.

    Article  CAS  PubMed  Google Scholar 

  3. Just T, Burgwald H, Broe MK. Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes. J Virol Methods. 1998;73:163–74.

    Article  CAS  PubMed  Google Scholar 

  4. Kimura H, Miyake K, Yamauchi Y, Nishiyama K, Iwata S, Iwatsuki K, et al. Identification of Epstein-Barr virus (EBV)-infected lymphocyte subtypes by flow cytometric in situ hybridization in EBV-associated lymphoproliferative diseases. J Infect Dis. 2009;200:1078–87.

    Article  CAS  PubMed  Google Scholar 

  5. Kawabe S, Ito Y, Gotoh K, Kojima S, Matsumoto K, Kinoshita T, et al. Application of flow cytometric in situ hybridization assay to Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases. Cancer Sci. 2012;103:1481–1448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fournier B, Boutboul D, Bruneau J, Miot C, Boulanger C, Malphettes M, et al. Rapid identification and characterization of infected cells in blood during chronic active Epstein-Barr virus infection. J Exp Med. 2020;217: e20192262.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ohga S, Ishimura M, Yoshimoto G, Miyamoto T, Takada H, Tanaka T, et al. Clonal origin of Epstein-Barr virus (EBV)-infected T/NK-cell subpopulations in EBV-positive T/NK-cell lymphoproliferative disorders of childhood. J Clin Virol. 2011;51:31–7.

    Article  CAS  PubMed  Google Scholar 

  8. Kanegane H, Wado T, Nunogami K, Seki H, Taniguchi N, Tosato G. Chronic persistent Epstein-Barr virus infection of natural killer cells and B cells associated with granular lymphocytes expansion. Br J Haematol. 1996;95:116–22.

    Article  CAS  PubMed  Google Scholar 

  9. Arai A. Advances in the study of chronic active Epstein-Barr virus infection: clinical features under the 2016 WHO classification and mechanisms of development. Front Pediatr. 2019;7:14.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Quintanilla-Martinez L, Swerdlow SH, Tousseyn T, Barrionuevo C, Nakamura S, Jaffe ES. New concepts in EBV-associated B, T, and NK cell lymphoproliferative disorders. Virchows Arch. 2023;482:227–44.

    Article  CAS  PubMed  Google Scholar 

  11. Meazza R, Tuberosa C, Cetica V, Falco M, Parolini S, Grieve S, et al. Diagnosing XLP1 in patients with hemophagocytic lymphohistiocytosis. J Allergy Clin Immunol. 2014;134:1381–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Henter JI, Horne A, Aricó M, Egeler RM, Filipovich AH, Imashuku S, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007;48:124–31.

    Article  PubMed  Google Scholar 

  13. Loren AW, Porter DL, Stadtmauer EA, Tsai DE. Post-transplant lymphoproliferative disorder: a review. Bone Marrow Transplant. 2003;31:145–55.

    Article  CAS  PubMed  Google Scholar 

  14. Kimura H, Morita M, Yabuta Y, Kuzushima K, Kato K, Kojima S, et al. Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J Clin Microbiol. 1999;37:132–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Watanabe Y, Sasahara Y, Satoh M, Looi CY, Katayama S, Suzuki T, et al. A case series of CAEBV of children and young adults treated with reduced-intensity conditioning and allogeneic bone marrow transplantation: a single-center study. Eur J Haematol. 2013;91:242–8.

    Article  PubMed  Google Scholar 

  16. Kimura H, Hoshino Y, Kanegane H, Tsuge I, Okamura T, Kawa K, et al. Clinical and virologic characteristics of chronic active Epstein-Barr virus infection. Blood. 2001;98:280–6.

    Article  CAS  PubMed  Google Scholar 

  17. Kimura H, Ito Y, Kawabe S, Gotoh K, Takahashi Y, Kojima S, et al. EBV-associated T/NK-cell lymphoproliferative diseases in nonimmunocompromised hosts: prospective analysis of 108 cases. Blood. 2012;119:673–86.

    Article  CAS  PubMed  Google Scholar 

  18. Okuno Y, Murata T, Sato Y, Muramatsu H, Ito Y, Watanabe T, et al. Defective Epstein-Barr virus in chronic active infection and haematological malignancy. Nat Microbiol. 2019;4:404–13.

    Article  CAS  PubMed  Google Scholar 

  19. J Wang M Su N Wei H Yan J Zhang Y Gong et al 2023 Chronic active Epstein-Barr virus disease originates from infected hematopoietic stem cells [published online ahead of print, 2023 Oct 12]. Blood blood.2023021074.

  20. Iwatsuki K, Miyake T, Hirai Y, Yamamoto T. Hydroa vacciniforme: a distinctive form of Epstein-Barr virus-associated T-cell lymphoproliferative disorders. Eur J Dermatol. 2019;29:21–8.

    Article  PubMed  Google Scholar 

  21. Toga A, Wada T, Sakakibara Y, Mase S, Araki R, Tone Y, et al. Clinical significance of cloned expansion and CD5 down-regulation in Epstein-Barr Virus (EBV)-infected CD8+ T lymphocytes in EBV-associated hemophagocytic lymphohistiocytosis. J Infect Dis. 2010;201:1923–32.

    Article  CAS  PubMed  Google Scholar 

  22. Yang X, Wada T, Imadome K, Nishida N, Mukai T, Fujiwara M, et al. Characterization of Epstein-Barr virus (EBV)-infected cells in EBV-associated hemophagocytic lymphohistiocytosis in two patients with X-linked lymphoproliferative syndrome type 1 and type 2. Herpesviridae. 2012;3:1.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Al-Mansour Z, Nelson BP, Evens AM. Post-transplant lymphoproliferative disease (PTLD): risk factors, diagnosis, and current treatment strategies. Curr Hematol Malig Rep. 2013;8:173–83.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Li Q, Spriggs MK, Kovats S, Turk SM, Comeau MR, Nepom B, et al. Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes. J Virol. 1997;71(6):4657–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the patients to participate in this study.

Funding

This work was supported by MEXT/JSPS KAKENHI (Grant Number: 22K07887) and Daiichi Sankyo Scholarship Donation Program to HK.

Author information

Author notes

  1. Dan Tomomasa, Kay Tanita and Yuriko Hiruma equally contribute to this study.

  2. Akihiro Hoshino and Hirokazu Kanegane contributed equally and share senior authorship.

Authors and Affiliations

  1. Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Dan Tomomasa, Kay Tanita & Tomohiro Morio

  2. Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, UMR 1163, INSERM, Imagine Institute, Paris, France

    Kay Tanita, Akihiro Hoshino, Benjamin Fornier & Sylvain Latour

  3. Faculty of Medicine, Tokyo Medical and Dental University (TMUD), Tokyo, Japan

    Yuriko Hiruma

  4. Deparment of Child Health and Development, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8519, Japan

    Akihiro Hoshino & Hirokazu Kanegane

  5. Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

    Ko Kudo

  6. Department of Hematology and Oncology, Shizuoka Children’s Hospital, Shizuoka, Japan

    Shohei Azumi

  7. Department of Pediatrics, Medical Research Institute KITANO HOSPITAL, PIIF Tazuke-Kofukai, Osaka, Japan

    Mitsutaka Shiota

  8. Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan

    Masayoshi Yamaoka

  9. Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Katsuhide Eguchi & Masataka Ishimura

  10. Department of Hematology, Saitama Medical Center, Saitama Medical University, Saitama, Japan

    Yuka Tanaka

  11. Department of Dermatology, Okayama University Graduate School of Medicine, Okayama, Japan

    Keiji Iwatsuki

  12. Division of Pediatrics and Perinatology, Faculty of Medicine, Tottori University, Yonago, Japan

    Keisuke Okuno

  13. Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Aichi Medical Center Nagoya First Hospital, Nagoya, Japan

    Asahito Hama

  14. Department of Pediatrics, Shiga University of Medical Science, Shiga, Japan

    Ken-Ichi Sakamoto & Takashi Taga

  15. Department of Hematology/Oncology, Osaka Women’s and Children’s Hospital, Osaka, Japan

    Kimitoshi Goto

  16. Department of Virology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

    Kimitoshi Goto

  17. Department of Pediatrics, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan

    Haruka Ota & Akihiro Ichiki

  18. Department of Pediatrics, Gifu Municipal Hospital, Gifu, Japan

    Kaori Kanda

  19. Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan

    Takako Miyamura

  20. Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan

    Saori Endo & Hidenori Ohnishi

  21. Department of Pediatrics, Tohoku University Graduate School of Medicine, Miyagi, Japan

    Yoji Sasahara

  22. Department of Hematology and Oncology, St. Marianna University School of Medicine, Kanagawa, Japan

    Ayako Arai

  23. Paediatric Haematology-Immunology and Rheumatology Unit, Necker Hospital, AP-HP.Centre - Université Paris Cité, Paris, France

    Benjamin Fornier

  24. Department for Advanced Medicine for Viral Infections, National Center for Child Health and Development, Tokyo, Japan

    Ken-Ichi Imadome

Authors
  1. Dan Tomomasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kay Tanita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuriko Hiruma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akihiro Hoshino
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ko Kudo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shohei Azumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mitsutaka Shiota
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Masayoshi Yamaoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Katsuhide Eguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Masataka Ishimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yuka Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Keiji Iwatsuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Keisuke Okuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Asahito Hama
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Ken-Ichi Sakamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Takashi Taga
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Kimitoshi Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Haruka Ota
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Akihiro Ichiki
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Kaori Kanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Takako Miyamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Saori Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Hidenori Ohnishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Yoji Sasahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Ayako Arai
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Benjamin Fornier
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Ken-Ichi Imadome
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Tomohiro Morio
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Sylvain Latour
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Hirokazu Kanegane
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

DT, Akihiro H and HK wrote the manuscript. DT, KT, and YH performed EBER-FISH. Ken-Ichi I performed beads sorting qPCR. Ko K, SA, MS, MY, KE, MI, YT, Keiji I, KO, Asahito H, KS, TT, KG, Haruka O, AI, Kaori K, Takako M, SE, Hidenori O, YS, and AA collected the patient data. BF, Tomohiro M and SL provided critical discussion. HK conceptualized the study. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Akihiro Hoshino or Hirokazu Kanegane.

Ethics declarations

Conflict of interests

All authors declare that they have no relevant conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 170 KB)

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tomomasa, D., Tanita, K., Hiruma, Y. et al. Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH. Int J Hematol (2024). https://doi.org/10.1007/s12185-024-03786-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12185-024-03786-0

Keywords

Search

Navigation