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.
Highlights
Sequence read that passed default quality control filters on the Illumina platform were aligned to Epstein-Barr virus (EBV) reference genome sequence followed by BLAST procedure against nucleotide database to determine the sequence identity. 91.99% (3,143,389) of the reads can be mapped to the reference genome indicating the high specificity of the amplicon generation
A position was defined to be homogeneous if the variant frequency is . = 95% and a position to be heterogeneous if the variant frequency is between 20% and 94%, both homogenous and heterogeneous positions require read depth to be 5 or above While we could not completely rule out artifactual sequence alterations that might occur during amplicon generation or during Illumina sequencing, we identified a total of 172 potential heterogeneous nucleotide positions in HKNPC, of which 143 were located in repeat sequences throughout the genome
Many studies aimed to identify nasopharyngeal carcinoma (NPC)-specific strains focused only on a limited number of genes expressed in NPC, predominantly Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), LMP2A, BZLF1, miR-BART, and Epstein-Barr virus–encoded small RNA 1 (EBER1) and -2
Summary
Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus infecting more than 90% of the world’s population and is associated with both non-malignant disease, such as infectious mononucleosis, as well as malignant diseases, such as nasopharyngeal carcinoma (NPC), endemic Burkitt’s lymphoma, Hodgkin’s disease, B- and T-cell lymphomas and rare cases of gastric carcinoma [1]. The EBV genome comprises approximately 170 kb and contains at least 86 open reading frames. The virus genome contains a long unique region interspersed by four major internal repeats (IR1 to IR4) and terminal repeats (TR). Nine latent proteins including Epstein-Barr nuclear antigen 1 (EBNA1), EBNA2, EBNA3A, -3B, -3C, EBNA-LP and latent membrane protein 1 (LMP1) and LMP2A, -2B are encoded by genes situated in the unique region of the genome [1]. Other open reading frames encode capsid proteins, transcription factors as well as lytic proteins of various functions [2]. In addition to protein-coding genes, EBV genome encodes non-coding EBV RNAs, such as Epstein-Barr virus–encoded small RNA 1 (EBER1) and 2 (EBER2), BART-derived microRNAs (miRNAs-BARTs) and BHRF1 microRNAs (miRNAs-BHRF1) [3,4]
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