Preliminary Study Of Epstein-Barr Virus Strain And Integration Mechanism Based On The Whole Genome Sequencing And Virus Capture Sequencing Technology

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpes virus and infects more than90%of the world’s population.The EBV genome is approximately171kb and contains at least86open reading frames. EBV is a key risk factor for many malignancy diseases such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL).The effects of the EBV oncoproteins on apoptosis and cell proliferation via interaction have been documented. Meanwhile, EBV DNA integration could cause host cell organization inflammation and tumorigenesis. It’s integration could induce the host genome instability and rearrangement. It’s could also cause the alterations of host transcriptome such as viral promoter driven human transcription, viral-human transcription fusion and form new fusion gene, and the host gene expression change.The mode and influence of EBV integration are still not clear due to the lack of an impartial method to detect and survey genome-wide EBV integration sites, although EBV integration into host genome has been reported.C666-1(NPC cell line) and Raji (BL cell line) are commonly studied EBV-positive cancer cells which have been analyzed in at least 3000publications in recent decades. A rare few EBV integrations sites in Raji were found in previous research by traditional methods. To deeply survey EBV integration in cancer genomes, we sequenced C666-1and Raji whole genomes by the next generation sequencing (NGS) technology and a total of909breakpoints were detected in the two cell lines.By using whole genome sequencing, we found large number of SNPs, InDels, SVs and CNVs both in C666-1and Raji (2,582,026SNPs,1038097InDels,5820SVs and1510CNVs in C666-1;2,805,619SNPs,542732InDels,6996SVs and426CNVs in Raji). Moreover, we observed most breakpoints located inside or nearby genome structural variations regions. It suggested that host genome instability provided an opportunity for EBV integration on one hand and the integration aggravated host genome instability on the other hand.The full-length sequence of C666-1EBV strain was submitted to the DNA Data Bank of Japan (DDBJ) database and assigned accession number AB828190. The full-length sequence of Raji EBV strain was also submitted to the DDBJ database and assigned accession number AB828191.Then, we respectively assembled the C666-1and Raji EBV strains which would be useful resources for EBV-relative studies. The last not the least, we constructed phylogenetic trees of eight EBV subtypes and the result showed that subtypes derived from the same disease were closely clustered, which indicated the high-accuracy of NGS and the correlation between subtypes and diseases.We also processed virus capture sequencing of18EBV associated NPC tumor biopsies to survey the EBV strain and integration mechanism in NPC.We found EBV integration in all the biospsies and EBV frequently integrated into the region of the host cell chromosome telomere and centromere.It suggested that the EBV could aggravate the tumorigenesis by integration of the host cell chromosome telomere and centromere region.We assembled the18EBV genome sequences of the NPC tumor biopsies and submitted the data to the DDBJ. We found the variations of the18virus genomes were mostly located in the virus genome95-100kb region and165-170region in comparison to the reference EBV sequence(EBV-WT). It suggested that the variations in the two regions may enhance the virus tumorigenesis in NPC.Sequence and phylogenetic analyses showed that by using the virus genome or the sequence of the EBNA1, subtypes derived from the same disease and geography were closely clustered. It indicated that the EBV genome and the EBNA1could be potential molecular markers in EBV associated diseases.Thus, we report the most comprehensive characterization of EBV strain and integration, and EBV shows the wide range and random integration to increase the tumorigenesis. The NGS provides an incomparable level of resolution on EBV integration and an convenient approach to obtain viral strain compared to any research technology before.