Identification and characterization of EBV genomes in spontaneously immortalized human peripheral blood B lymphocytes by NGS technology

Abstract

BackgroundWe conducted genomic sequencing to identify Epstein Barr Virus (EBV) genomes in 2 human peripheral blood B lymphocytes that underwent spontaneous immortalization promoted by mycoplasma infections in culture, using the high-throughput sequencing (HTS) Illumina MiSeq platform. The purpose of this study was to examine if rapid detection and characterization of a viral agent could be effectively achieved by HTS using a platform that has become readily available in general biology laboratories.ResultsRaw read sequences, averaging 175 bps in length, were mapped with DNA databases of human, bacteria, fungi and virus genomes using the CLC Genomics Workbench bioinformatics tool. Overall 37,757 out of 49,520,834 total reads in one lymphocyte line (# K4413-Mi) and 28,178 out of 45,335,960 reads in the other lymphocyte line (# K4123-Mi) were identified as EBV sequences. The two EBV genomes with estimated 35.22-fold and 31.06-fold sequence coverage respectively, designated K4413-Mi EBV and K4123-Mi EBV (GenBank accession number KC440852 and KC440851 respectively), are characteristic of type-1 EBV.ConclusionsSequence comparison and phylogenetic analysis among K4413-Mi EBV, K4123-Mi EBV and the EBV genomes previously reported to GenBank as well as the NA12878 EBV genome assembled from database of the 1000 Genome Project showed that these 2 EBVs are most closely related to B95-8, an EBV previously isolated from a patient with infectious mononucleosis and WT-EBV. They are less similar to EBVs associated with nasopharyngeal carcinoma (NPC) from Hong Kong and China as well as the Akata strain of a case of Burkitt’s lymphoma from Japan. They are most different from type 2 EBV found in Western African Burkitt’s lymphoma.

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

“…A consensus EBV genomic sequence of C666-1 was recently constructed by reference mapping (19). Most recently, two more EBV genomes in immortalized human B lymphocyte cell lines were sequenced using the Illumina MiSeq platform (20). Sequencing reads from total DNA of the cell lines were mapped to the EBV reference genome, and the mappable reads were assembled to yield the two EBV genomes, K4413-Mi and K4123-Mi.…”

Genome-wide Analysis of Epstein-Barr Virus (EBV) Integration and Strain in C666-1 and Raji Cells

“…The MAFFT [37] software was used to align the EBV genomes of C666-1 (AB828190) and Raji (AB828191) with other 18 strains reported previously: EBV-WT (NC007605) [34], B95-8 (V01555) [34], AG876 (DQ279927) [38], Akata (KC207813) [39], Mutu (KC207814) [39], K4123-Mi (KC440851) [40], K4413-Mi (KC440852) [40], GD1 (AY961628) [41], GD2 (HQ020558) [42], HKNPC1(JQ009376) [43], and HKNPC2 ~ HKNPC9 (KF992564 ~ KF992571) [44]. Molecular Evolutionary Genetics Analysis (MEGA) [45] software, version 5.2 was used to perform the Phylogenetic analyses by Neighbor-joining (NJ) algorithm, based on multiple sequence alignments of the eight viral genomes and encoded genes.…”

Epstein–Barr virus strains and variations: Geographic or disease‐specific variants?

“…B95.8 strain (GenBank: V01555) genome was first sequenced in 1984 [Baer et al, ] and updated in 2003 [de Jesus et al, ]; while AG876 genome (GenBank: DQ279927) was published in 2006 [Dolan et al, ]. Furthermore, other EBV strains were isolated and fully sequenced in the past 10 years: GD1, isolated from the saliva of a NPC patient in (GenBank: AY961628) [Zeng et al, ]; GD2, isolated from a biopsy of a NPC patient (GenBank: HQ020558) [Liu et al, ]; M81, isolated from a NPC patient (GenBank: KF373730) [Tsai et al, ]; HKNPC1, isolated from a NPC patient (GenBank: JQ009376) [Kwok et al, ]; C666‐1, isolated from the NPC cell line (GenBank: KC617875) [Tso et al, ]; K4123‐Mi (GenBank: KC440852) and K4413‐Mi (GenBank: KC440851), isolated from spontaneous lymphoblastoid cell lines (LCLs) [Lei et al, ]; African Mutu BL (GenBank: KC207814) and Japanese Akata (GenBank: KC207813) cell lines [Lin et al, ]. The advances in sequencing technologies have allowed the fast evolution and knowledge on the genome of EBV strains [Farrell, ].…”

“…The advances in sequencing technologies have allowed the fast evolution and knowledge on the genome of EBV strains [Farrell, ]. Indeed, recent studies have started to fully sequence EBV samples from different diseases and locations to characterize its geographic variation and disease association [Depledge et al, ; Lei et al, ; Kwok et al, ; Feederle et al, ; Palser et al, ].…”