Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples

Abstract

Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis.

Importance: This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

FIG 1

Workflow of sequencing and analysis of EBV genomes. Input DNA extracted from primary biopsy specimens of NPC or cell lines go through library extraction, EBV DNA capture, index tagging, and next-generation sequencing to generate sequencing reads. Reads mappable to EBV go through filtering and trimming processes and were de novo assembled to determine contigs. The contigs were aligned to the reference to generate scaffolds, and the contigs were joined by Sanger sequencing. Subsequent analyses are performed on the resulting EBV genomes.

FIG 2

Genetic variations among NPC-EBV strains. (A) Mutations of EBV strains isolated from NPC relative to the reference EBV strain (NC_007605). Mutations in internal repeats and terminal repeats are disregarded, and the regions are shaded in gray. (B) Mutations of EBV strains isolated from NPC relative to HKNPC1. Rightward and leftward open reading frames of EBV are overlaid on top of the mutations.

FIG 3

Nonsynonymous mutations of HKNPC2 to -9. (A) Number of nonsynonymous mutations contained in the nine categories of EBV-encoded proteins. The majority of the amino acid changes are located in latent proteins (blue) in all of the HKNPC strains, followed by tegument (red) and membrane protein (green). (B) Amino acid changes in CD8⁺ and CD4⁺ specific T cell epitopes. Amino acid changes in at least one of the HKNPC strains at CD8⁺ and CD4⁺ specific T cell epitopes are marked with solid and hollow arrows, respectively. Stacking arrows indicate that the amino acid change is in a peptide which serves as both CD8⁺ and CD4⁺ epitopes. X, nonsense mutation causing a truncation of BLLF1 at QNP epitope; Ins, insertion at EBNA2.

FIG 4

Phylogenetic analysis on whole EBV genomes and protein-encoding nucleotide sequences of LMP-1, BLLF1, and BPLF1 genes. The layout of phylogenetic trees generally follows the geographical origins of the EBV strains. The only type 2 sequence included in the analysis, AG876, is indicated as most distant from other strains in whole-genome and BLLF1 sequences. Analysis on whole-genome sequences for BLLF1 and BPLF1 shows that HKNPC6 and -7 are distinct from other HKNPC strains. All analyses are rooted using rhesus lymphocryptovirus. The numbers at the internal nodes correspond to the bootstrap values, obtained in an analysis of 500 replicates.

FIG 5

Recombination analysis of HKNPC2. (A) BootScan profile of HKNPC7 (red) and -9 (blue) against HKNPC2. HKNPC7 has a higher percentage of permutated trees in regions flanking internal repeat 2 (IR2), indicating a recombination event. (B) Closeup view of regions flanking IR2. (C) Similarity plot generated by SimPlot. Both HKNPC7 and -9 are highly similar to HKNPC2, with a slight drop of similarity of HKNPC9 in the region of recombination. (D) Recombination model of HKNPC2. (E to G) Neighbor-joining trees of regions of and flanking the recombinant fragments.