*Purpose: The effect of the genetic diversity of the Epstein-Barr virus (EBV) on clinical outcomes in the transplant setting is important to study. We examined the phylogenetic relationship between the Epstein-Barr virus nuclear antigen-1 (EBNA1) strains from within and outside of Canada that have been associated with PTLD in an effort to identify EBNA1 variants that are associated with PTLD.

*Methods: We examined the variants of the EBNA1 gene among (N=44) patients enrolled in a Canadian pediatric multi-centre study of patients (<18 years old) with and without EBV-related post-transplant lymphoproliferative disorder (PTLD) (CNTRP POSITIVE Study). The EBNA1 gene was sequenced from blood samples during EBV DNAemia. Publicly available PTLD sequences and sequences from Canadian patients with infectious mononucleosis (IM) were used for comparison. Sequencing was done by dideoxy DNA sequencing methodology and the sequences were aligned with a reference strain of EBV (B95-8). EBNA1 variants were classified into prototype (P) and variant (V) strains, based on the amino acid change that occurred at codon 487 (P-ala, P-thr, V-leu, V-val, V-pro).

*Results: We identified 54 EBNA1 sequences in the Canadian cohort and 10 sequences from Australia and USA. Twenty-one were from solid organ transplants (SOT) recipients of which 11 developed PTLD. There were 13 patients who had bone marrow transplantation (BMT) of which 3 developed PTLD. The transplant patient samples were compared to 10 IM samples (patients less than 24 years of age). The predominant EBNA1 subtypes were: Canada – BMT, P-thr, 84.6%; Canada – SOT, P-thr, 71.4%; Canada – IM, P-thr, 80%; Australia, P-thr, 75%; USA, V-leu, 100%. The majority of sequences fell into two main clades. The strains from Canada, including those from patients with and without PTLD clustered along with external strains with no clear evidence of geographic correlation. Sequences that were P-thr and V-leu as well as P-ala and V-val were linked with specific mutation patterns that were previously reported in the literature to likely be of functional significance.

*Conclusions: Analyses based on the EBNA1 gene show phylogenetic similarities between EBV strains from Canadian transplant patients and patients with infectious mononucleosis. These strains were phylogenetically related to strains from other geographic regions. Our results also suggest that changes at codon 487 may be linked with other substitutions within the gene that are likely of functional relevance based on the published literature.

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