*Purpose: Epstein Barr virus (EBV) is a gamma herpes virus that is associated with a variety of human malignancies including EBV+ B cell lymphomas in the setting of post-transplant lymphoproliferative disorder (PTLD). The development of EBV+ B cell lymphomas in PTLD is generally attributed to the unintended impact of immunosuppression on the ability of EBV-specific T cells to control the virus. However, diversity within the EBV genome may also alter the immune response to EBV or the oncogenic potential of the virus. The purpose of this study is to determine the diversity of the EBV genome in transplant recipients with EBV+ PTLD, and EBV seropositive transplant recipients, and to identify variations in the EBV genome that are associated with EBV+ PTLD.

*Methods: DNA was isolated from whole blood of transplant recipients (n=12) with either low (<1000 copies/υg DNA) or high (>1000 copies/υg DNA) levels of EBV, or with biopsy confirmed EBV+ PTLD (n-=6). DNA was also isolated from EBV+ B cell lines (n=6) generated from the blood of transplant recipients with EBV+ PTLD. The EBV genome was amplified by PCR using primers targeting non-repetitive regions and amplicons generated for whole genome sequencing. Fisher’s exact test with CLC Genomics Workbench was used to determine which variations were associated with the PTLD group compared to the EBV-High and EBV-Low groups. NetMHC and NetMHCII were used to predict the impact of PTLD variations on binding of T cell epitopes to HLA alleles.

*Results: A total of 97 x 10⁶ sequence reads of 150-base pair-end reads were generated from the combined pool of amplicons, with an average of 3 x 10⁶ sequence reads per sample. Hotspots of variation (insertions, deletions, single nucleotide changes, or multiple sequential nucleotide variations) in EBV genomes from subjects with PTLD were identified and were increased in latent cycle genes. The EBNA3C gene had significantly more non-synonymous variations in PTLD-derived genomes compared to genomes isolated from transplant recipients without PTLD. Fifteen non-synonymous variations, including 8 in EBNA3C, were associated with the development of PTLD. Six of these variations (40%) had a predicted reduction, or loss of binding to the corresponding HLA class I or class II molecule.

*Conclusions: These findings indicate that there is extensive diversity in EBV genomes isolated from transplant recipients, and that PTLD-associated variations in EBV can be identified. Moreover, these variations may impact the ability of recipient T cells to recognize and respond to EBV-infected B cells, and could contribute to the pathogenesis of EBV+ PTLD.

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