Direct Sequencing and Characterization of a Clinical Isolate of Epstein-Barr Virus from Nasopharyngeal Carcinoma Tissue by Using Next-Generation Sequencing Technology

Abstract

Epstein-Barr virus (EBV)-encoded molecules have been detected in the tumor tissues of several cancers, including nasopharyngeal carcinoma (NPC), suggesting that EBV plays an important role in tumorigenesis.However, the nature of EBV with respect to genome width in vivo and whether EBV undergoes clonal expansion in the tumor tissues are still poorly understood. In this study, next-generation sequencing (NGS) was used to sequence DNA extracted directly from the tumor tissue of a patient with NPC. Apart from the human sequences, a clinically isolated EBV genome 164.7 kb in size was successfully assembled and named GD2 (GenBank accession number HQ020558). Sequence and phylogenetic analyses showed that GD2 was closely related to GD1, a previously assembled variant derived from a patient with NPC. GD2 contains the most prevalent EBV variants reported in Cantonese patients with NPC, suggesting that it might be the prevalent strain in this population. Furthermore, GD2 could be grouped into a single subtype according to common classification criteria and contains only 6 heterozygous point mutations, suggesting the monoclonal expansion of GD2 in NPC. This study represents the first genome-wide analysis of a clinical isolate of EBV directly extracted from NPC tissue. Our study reveals that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV in vivo. The pipeline could also be applied to the study of other pathogen-related malignancies. With additional NGS studies of NPC, it might be possible to uncover the potential causative EBV variant involved in NPC.Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects more than 90% of the worldwide human population. Following the first discovery of EBV particles in cultured lymphoma cells from patients with Burkitt's lymphoma (BL) (12, 13), EBV and its encoded molecules were also detected in cases of nasopharyngeal carcinoma (NPC) (52) and several other malignancies, such as non-Hodgkin lymphoma (NHL) (51) and T-cell and Hodgkin lymphomas (22,43,44). Both the presence of virus sequences in tumor cells and the virus's oncogenic potency (24, 46) strongly associate EBV with NPC. However, the genome-wide nature of the EBV in NPC tissue is still poorly understood.EBV harbors different genetic variations in different geographic populations (4,10,18,21,40,(48)(49)(50). Likewise, the prevalence characteristics of NPC show remarkable geographical and ethnic differences, with a high prevalence rate in southern China, especially in the Guangdong province (5). Several EBV genes, including EBNA2, LMP1, and EBNA1, have been implicated in the development of NPC (10,17,28,34). Certain EBV subtypes such as China 1 and V-val, as classified by the sequence variations of these genes, were found more frequently in patients with NPC than in controls and thus have been associated with NPC (26, 50). However, an NPCspecific EBV subtype has not yet been identified, suggesting that EBV subtypes cannot be classi...

Discovery of recurrent structural variants in nasopharyngeal carcinoma

“…Consistent with the involvement of Epstein-Barr Virus (EBV) in NPC etiology and previous estimates of 1-30 viral genome copies per cell (Nanbo et al 2007;Liu et al 2011), we detected large amounts of EBV genomic sequence in NPC-5989 but not in its matched normal sample (1.86 million versus 521 read pairs). We estimate that there were about 108 EBV genome copies per cell in the tumor sample (Supplemental Material).…”

Whole-Genome Sequencing of the Akata and Mutu Epstein-Barr Virus Strains

“…For example, Liu et al (16) used at least 24 paired-end lanes of Illumina sequence data to obtain 17ϫ coverage of an EBV genome. To overcome this obstacle, we first took advantage of EBV's lytic replication cycle to amplify viral genomic DNA and increase the ratio of viral genome to cellular genome.…”

Genomic Diversity of Epstein-Barr Virus Genomes Isolated from Primary Nasopharyngeal Carcinoma Biopsy Samples

“…The EBV DNA was PCR amplified, subcloned, and sequenced by Sanger sequencing. Both GD2 and HKNPC1 were direct isolates from primary NPC biopsy specimens (22). GD2 was obtained as a small subset of sequence data from next-generation sequencing of the total DNA sequences derived from a NPC biopsy specimen (22).…”

“…Both GD2 and HKNPC1 were direct isolates from primary NPC biopsy specimens (22). GD2 was obtained as a small subset of sequence data from next-generation sequencing of the total DNA sequences derived from a NPC biopsy specimen (22). HKNPC1 was sequenced from a primary NPC biopsy specimen by next-generation sequencing after enrichment of EBV DNA by PCR amplification (23).…”