Sequence analysis of EBV DNA isolated from mouth washings and PBMCs of healthy individuals and blood of EBV-LPD patients

BACKGROUND: Lymphoproliferative disorder (LPD) caused by Epstein-Barr virus (EBV) is a severe complication of bone marrow transplantation. The EBV strain causing LPD is of either donor or recipient origin, however, available data are limited to only a small number of cases. To obtain solid evidence, comparison of the EBV strain that caused the EBV-LPD with pre-stem cell transplantation (SCT) EBV strains of donor and recipient is imperative. Available techniques rely on the production of EBV transformed lymphoblastoid cell lines and lack sensitivity.

OBJECTIVE: The aim of this study was to develop a simple method for EBV sequence analysis on mouth washings (MWs) and peripheral blood mononuclear cells (PBMCs).

STUDY DESIGN: EBV DNA was extracted from MWs and PBMCs that were collected from 20 healthy individuals. DNA was used for sequence analysis, using a polymerase chain reaction for the C-terminus of the LMP-1 gene.

RESULTS: In seropositive individuals EBV DNA could be detected in 11/14 (79%) MWs and in 13/14 (93%) PBMC samples. Sequence analysis showed that in 11 out of 14 (79%) healthy individuals sequence patterns could be established. In these 11 healthy individuals 13 sequence patterns could be detected. Eleven of these 13 patterns (84.6%) were unique. These results encouraged us to explore the feasibility of this method on EBV DNA isolated from plasma from 9 EBV-LPD patients at time of EBV reactivation. In 7 EBV-LPD patients 8 sequence patterns were detected. Six out of 8 sequence patterns (75%) were unique.

CONCLUSION: Our method is suitable for strain identification and we intend to use this technique to evaluate EBV origin in EBV-LPD patients.