Genome-Wide Analysis of 18 Epstein-Barr Viruses Isolated from Primary Nasopharyngeal Carcinoma Biopsy Specimens

Abstract

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that is highly prevalent in almost all human populations and is associated with many human cancers, such as nasopharyngeal carcinoma (NPC), Hodgkin's disease, and gastric carcinoma. However, in these EBV-associated cancers, only NPC exhibits remarkable ethnic and geographic distribution. We hypothesized that EBV genomic variations might contribute to the pathogenesis of different human cancers in different geographic areas. In this study, we collected 18 NPC biopsy specimens from the Hunan Province in southern China and de novo assembled 18 NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HN1 to HN18. This was achieved through target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, to reveal sequence diversity. These EBV genomes harbored 20,570 variations totally, including 20,328 substitutions, 88 insertions, and 154 deletions, compared to the EBV reference genome. Phylogenetic analysis revealed that all NPC-EBV genomes were distinct from other EBV genomes. Furthermore, HN1 to HN18 had some nonsynonymous variations in EBV genes including genes encoding latent, early lytic, and tegument proteins, such as substitutions within transmembrane domains 1 and 3 of LMP1, FoP_duplication, and zf-AD domains of ENBA1, in addition to aberrations in noncoding regions, especially in BamHI A rightward transcript microRNAs. These variations might have potential biological significance. In conclusion, we reported a genome-wide view of sequence variation in EBV isolated from primary NPC biopsy specimens obtained from the Hunan Province. This might contribute to further understanding of how genomic variations contribute to carcinogenesis, which would impact the treatment of EBV-associated cancer.IMPORTANCE Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV) infection and exhibits remarkable ethnic and geographic distribution. Hunan Province in southern China has a high incidence rate of NPCs. Here, we report 18 novel EBV genome sequences from viruses isolated from primary NPC biopsy specimens in this region, revealing whole-genome sequence diversity.

Keywords: EBV; Epstein-Barr virus; NPC; genetic variations; nasopharyngeal carcinoma; phylogenetic analysis; virus capture sequencing.

FIG 1

Distribution of nonsynonymous variations in 18 NPC biopsy-derived Epstein-Barr virus (NPC-EBV) genomes. Genome sequences of 18 NPC-EBVs, designated HN1 to HN18, were assembled and compared to the reference EBV sequence (NC007605). Circos plots representing the nonsynonymous variations in HN1 to HN9 (A) and HN10 to HN18 (B) tracks from outer to inner represent the following: track 1 (outermost), coding exons for latent genes (red), lytic genes (blue), and RPMS1/miRNAs (green); track 2, repeat and regulatory elements (black); and track 3, nonsynonymous variations (bright green) in the NPC-EBV genomes compared to the EBV reference genome sequence. (C) Genome-wide distribution of single-nucleotide variation (SNVs) in HN1 to HN18. The genomic positions are related to the EBV reference sequence (NC007605). SNVs were identified by cross_match and BWA software. The highest incidences of variations were found at 95 to 105 kb (where the EBNA1 and BKRF1 genes are located) and 165 to 172 kb (which includes the LMP1 gene). (See File S1 in the supplemental material for an enlargeable version of the figure.)

FIG 2

Categories of nonsynonymous variations in 18 NPC-EBVs. Nonsynonymous variations in the HN1 to HN18 samples were categorized based on the functions of EBV-encoded proteins. Most amino acid changes were located in latency-associated proteins (red), followed by tegument-associated proteins (light blue) and membrane-associated proteins (green).

FIG 3

Phylogenetic analyses of 18 EBV genomes assembled in this study and other EBV strains based on whole EBV genomic sequences. The numbers at the internal nodes correspond to bootstrap values obtained based on the analysis of 500 replicates.

FIG 4

Phylogenetic analysis of some important EBV coding genes. Sequences of LMP1 (A), BZLF1 (B), EBNA1 (C), BKRF2 (D), BKRF3 (E), and BKRF4 (F) genes derived from this study and those from previously reported EBV genomes were analyzed. The numbers at the internal nodes correspond to the bootstrap values obtained based on the analysis of 500 replicates.

FIG 5

Sequence variations in the amino acid sequences of important EBV proteins. Alignment of partial protein sequences of LMP1 (A), EBNA1 (B), BKRF2 (C), and BKRF3 (D) based on 18 EBV genomes assembled in this study and those of other EBV strains. Variant positions are marked in yellow. Functional domains in this protein are indicated above the consensus sequence. (See File S1 in the supplemental material for an enlargeable version of the figure.)

FIG 6

Variations in EBV-encoded miRNAs. Alignment of pre-miRNA sequences of EBV-encoded miRNAs BART19 (A), BART10 (B), BART 17 (C), and BART 8 (D) from 18 EBV genomes assembled in this study and those of other EBV strains. Variant positions are marked in red; mature miRNA sequences are marked above the consensus sequence. (See File S1 in the supplemental material for an enlargeable version of the figure.)

FIG 7

Workflow for sequencing and analysis of EBV genomes from NPC biopsy specimens.