Purpose: In transplant recipients, Epstein-Barr Virus (EBV) is associated with the development of B cell lymphomas in post-transplant lymphoproliferative disorder (PTLD). We used next-generation sequencing to characterize the diversity of the EBV genome in PTLD and identify novel mutations that may play role in tumorigenesis.

Methods: DNA was isolated from 6 EBV+ spontaneous lymphoblastoid B cell lines (sLCL) derived from PTLD patients, and a set of 59 overlapping primers was used to amplify the EBV genome. The products were gel-extracted, normalized to equal molecular quantity and pooled. The pooled samples were prepared for sequencing using KAPA DNA library preparation kit, barcoded, and run on Illumina MiSeq. Reads were trimmed, filtered using a quality score of 20, mapped to the reference AJ507799 EBV genome, and assembled using Genome Analysis Toolkit (GATK) and Burrow Wheeler Aligner. Single nucleotide variations (SNVs), insertions and deletions (indels) were determined using GATK. A phylogenetic analysis was done using CLC Genomics Workbench.

Results: The EBV genomes from all 6 sLCLs (sLCL1-6) were successfully sequenced. The coverage of the aligned reads to the reference genome ranged from 73 to 95% with an average of 85%. The mean read depth for all 6 genomes was 6,553 and the mean GC content was 59%. The total number of variations for all sLCLs was 2,448. There were 1,574 substitutions and 75 indels in the coding regions and 448 substitutions, 112 indels in the non-coding regions of the genome. Within the coding regions, there were a total of 634 non-synonymous variations for sLCL1-6. Also, sLCL1-6 shared 63 variations: 59 SNVs and 4 indels. The latent cycle genes were the most diverse genes, containing 121 non-synonymous mutations that resulted in amino acid changes. The remaining non-synonymous mutations were in lytic proteins of the virus. Phylogenetic analysis of sLCL1-6 and 17 published EBV genomes from non-PTLD genomes, including 4 EBV genomes derived from healthy individuals, revealed that the 6 sLCL genomes formed a clade, clustering closest to each other.

Conclusions: The EBV genome is highly diverse, especially the viral latent cycle genes, which are responsible for proliferation of the host B cell. Moreover, EBV genomic diversity in PTLD is distinct from EBV genomic diversity of non-PTLD specimens. Thus, we have identified novel EBV mutations that may play an important role in tumorigenesis or immune evasion, contributing to PTLD pathogenesis.

CITATION INFORMATION: Maloney E, Martinez O. Genomic Diversity of Epstein-Barr Virus in Post-Transplant Lymphoproliferative Disorder. Am J Transplant. 2017;17 (suppl 3).

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