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).


METHODS
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.


CONCLUSIONS
Our method is suitable for strain identification and we intend to use this technique to evaluate EBV origin in EBV-LPD patients.
van Kooij
B
B
Departments of Virology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
Thijsen
S F T
SF
Meijer
E
E
Niesters
H G M
HG
van Esser
J W J
JW
Cornelissen
J J
JJ
Verdonck
L F
LF
van Loon
A M
AM
D000328:Adult; D004279:DNA, Viral / Q000032:analysis*; D020031:Epstein-Barr Virus Infections / Q000097:blood / Q000175:diagnosis*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017708:Leukocyte Transfusion / Q000009:adverse effects; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008232:Lymphoproliferative Disorders / Q000097:blood / Q000209:etiology / Q000821:virology*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D004279:DNA, Viral