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Clinical Trial
. 2012;7(5):e36939.
doi: 10.1371/journal.pone.0036939. Epub 2012 May 10.

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated From Primary Nasopharyngeal Carcinoma Biopsy

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Free PMC article
Clinical Trial

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated From Primary Nasopharyngeal Carcinoma Biopsy

Hin Kwok et al. PLoS One. .
Free PMC article

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, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8(+) T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Circular representation of HKNPC1 genome.
Numbered tracks represent the following: (1) PCR amplicons prepared for next-generation sequencing (NGS); (2) reverse open reading frames; (3) forward open reading frames; (4) repeat regions; (5) read depth of NGS reads, height of the track represents 10,000 reads; single nucleotide variations (SNVs) and indels of (6) HKNPC1, (7) GD1, (8) GD2, and (9) AG876, in comparison to reference EBV sequence. This figure was created using Circos software .
Figure 2
Figure 2. Comparison of HKNPC1 genome to other EBV genomes.
(A) Genome-wide distribution of SNVs of HKNPC1. Coordinates of SNVs were extracted by cross_match software and a density plot was constructed using 500-bp non-overlapping windows. (B) Similarity graphs of reference EBV, GD1, GD2 and AG876 compared against HKNPC1. The four genomes generally have high sequence identity with HKNPC1, except repeat regions (brown) and polymorphic genes (blue). EBNA2, EBNA3A, -3B, and -3C show lower identity in AG876 (type 2), than the other three type 1 EBV genomes. The figure was generated by mVISTA software, using 100-bp moving window with minimum identity of 70% and maximum identity of 100%.
Figure 3
Figure 3. Phylogenetic analysis of the five EBV genomes.
Phylogenetic tree based on DNA and protein sequences of the five EBV strains. (A) DNA sequence of complete genome of the five strains, with poorly aligned and highly divergent sequences masked by Gblocks. Phylogenetic trees based on protein sequence alignment of (B) BZLF1, (C) LMP1, and (D) EBNA1 were generated. All these trees showed a closer distance among the three NPC-related EBV strains, GD1, GD2, and HKNPC1, than the other two viral strains. Phylogenetic analysis was performed using MEGA software (version 5), by Neighbor-joining (NJ) algorithm. Divergence scale, in numbers of substitution per site, is shown under each tree.
Figure 4
Figure 4. Summary of single nucleotide variations and non-synonymous mutations in GD1, GD2 and HKNPC1 genomes.
(A) Number of single nucleotide variants (SNVs) of HKNPC1, GD1, GD2 and AG876 with each genome compared against reference EBV. The orange region represents the 322 SNVs shared by GD1, GD2 and HKNPC1 with exclusion of those of AG876, whereas the blue region represents the 347 SNVs shared by GD2 and HKNPC1 with exclusion of those of AG876 and GD1. (B) Non-synonymous SNVs shared by GD1, GD2 and HKNPC1 were categorized by protein function based on the work of Tabouriech et al. . (C) Non-synonymous SNVs shared by GD1, GD2 and HKNPC1 and those shared by GD2 and HKNPC1 were pooled together and categorized by function of the genes.
Figure 5
Figure 5. Location of amino acid changes of EBV proteins encoded by GD1, GD2 and HKNPC1 genomes.
Amino acids changes in EBV proteins with known or putative function due to non-synonymous SNVs are marked by arrows. Red arrows indicate amino acid changes shared by HKNPC1, GD1 and GD2, but not in AG876. Blue arrows indicate amino acid changes shared by HKNPC1 and GD2, but not in GD1 and AG876. Known or predicted cytoplasmic domain (brown), transmembrane domain (green) and extracellular domain (yellow) of membrane proteins are illustrated. Black bars represent specialized features of BALF4 (glycoprotein B), EBNA1, LMP1, EBNA1 and BPLF1.

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