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The sequence analysis of Epstein–Barr virus EBNA1 gene: could viral screening markers for nasopharyngeal carcinoma be identified?


Epstein–Barr virus (EBV) has been identified as a group 1 carcinogenic agent, particularly for nasopharyngeal carcinoma (NPC). The sequence diversity of EBV nuclear antigen 1 (EBNA1) reflects region-restricted polymorphisms, which may be associated with the development of certain malignancies. The aims of the present study were to evaluate EBV EBNA1 gene polymorphisms circulating in NPC, infectious mononucleosis, and isolates from patients with transplanted organs to determine if EBNA1 sequence specificities are useful as viral biomarkers for NPC. Forty biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), 31 plasma samples from patients with mononucleosis syndrome, and 16 plasma samples from patients after renal transplantation were tested in this study. The EBNA1 gene was amplified by nested PCR. Further investigation included sequencing, phylogenetic, and statistical evaluations. Eighty-seven sequences were identified as one of the four EBNA1 subtypes, P-Ala, P-Thr, V-Val, and V-Ala, with further classification into ten subvariants. Of these, P-Thr-sv-1 and P-Thr-sv-3 have never been identified in Europe, while V-Val-sv-1 was newly discovered. Statistical analysis revealed significant differences in the distribution of EBNA1 P-Thr subvariants between the three groups of patients, with noticeable clustering of P-Thr-sv-5 in NPC isolates (p < 0.001). EBV EBNA1 showed no sequence specificity in primary infection. This research revealed a newly discovered EBNA1 subvariant. Importantly, EBNA1 P-Thr-sv-5 showed carcinoma-specific EBNA1 variability. Thus, identification of this subvariant should be considered as a viral screening marker for NPC or UCNT.

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The authors are grateful to the laboratory technicians Gabrijela Pavlovic and Marija Zivotic from the Virology Department, Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade for their technical assistance.


This study was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Research Grant no. 175073.

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Correspondence to Ana V. Banko.

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The authors declare that they have no conflict of interest.

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All procedures performed in study involving human participants were in accordance with the ethical standards of the Ethics Committee of Faculty of Medicine, University of Belgrade no. 29/VI-12 and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For retrospective part of this study, formal consent was not reqired.

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Banko, A.V., Lazarevic, I.B., Karalic, D.Z. et al. The sequence analysis of Epstein–Barr virus EBNA1 gene: could viral screening markers for nasopharyngeal carcinoma be identified?. Med Microbiol Immunol 208, 81–88 (2019).

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  • Epstein–Barr virus (EBV)
  • EBNA1
  • Nasopharyngeal carcinoma
  • Sequence variability
  • Mononucleosis