Sequence analysis of epstein-barr virus genomes in nasopharyngeal carcinoma

Abstract

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, we aimed to sequence the complete EBV genomes harbored in NPC tumor biopsies and compare against the non-NPC EBV strains to identify NPC-specific EBV variations.

In the first part of the study, EBV genome contained in one primary NPC tumor biopsy was PCR-amplified and sequenced using next-generation and dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Accession number JQ009376), was generated by reference mapping and it appears to be a uniform strain in general despite minor heterogeneity. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC strains. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels). We found 76 non-synonymous SNVs shared amongst the Chinese GD1, GD2 and HKNPC1 isolates, while another 88 nonsynonymous SNVs were shared only by the two NPC tumor-derived strains HKNPC1 and GD2.

In the second part of the study, SureSelect target enrichment technology was used instead of PCR to capture EBV DNA from total DNA. The study was scaled-up to sequence EBV strains in cell lines, saliva and NPC tumor, using the MiSeq Personal Sequencer and the Genome Analyzer IIx platforms. The reads were de novo assembled to generate 17 complete EBV genomes, out of which 9 were NPC-EBV strains. Phylogenetic analysis of all available EBV strains has demonstrated that all NPC strains were type 1 EBV. Phylogeny predicted by LMP-1 gene showed clear geographical pattern of where the EBV strains were isolated. A total of 5,011 variations were identified by comparing every EBV strain against the reference. MicroRNAs and EBERs are generally well conserved across all genomes. Comparative analysis of variations between NPC and non-NPC EBV strains discovered 904 NPC-specific variations, out of which 112 appeared in more than one NPC strains. Among these recurrent variations, 39 non-synonymous substitutions and seven deletions in coding region were found. About half of these recurrent variations were located in EBNA-3A, -3B and -3C, while the rest was found in latent, tegument, capsid and packaging-related proteins and transcription factors. There were two NPC EBV strains isolated from the primary tumors which later diagnosed to have distant metastasis. Unique variations were shared in these two EBV strains in regions between IR2 and IR3, where genes such as BPLF1, BOLF1 and EBNA-3A, -3B and -3C were located, and leftward of IR3, where BBLF2/3 and BBRF1 were found. In conclusion, we have demonstrated the feasibility of target capture and next-generation sequencing in whole genome sequencing of EBV. Comparison of reference mapping and de novo assembly of EBV sequences illustrated that both are feasible approaches, though de novo assembly is preferred since the method is less dependent on the reference genome. Large-scale sequencing of NPC and non-NPC EBV strains may facilitate the discovery of previously unknown variations of biological significance and reveal the diverse role of EBV in NPC pathogenesis. words)