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

Lin, Zhen; Wang, Xia; Strong, Michael J.; Concha, Monica; Baddoo, Melody; Xu, Guiyin; Baribault, Carl; Fewell, Claire; Hulme, William; Hedges, Dale J.; Taylor, Christopher M.; Flemington, Erik K.

doi:10.1128/jvi.02517-12

Cited by 101 publications

(114 citation statements)

References 29 publications

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“…AG876 was originated from a Ghanaian case of Burkitt's lym- (17). C666-1 is a subclone of C666, an epithelial cell line derived from an NPC xenograft of southern Chinese origin (18).…”

Section: Importancementioning

confidence: 99%

“…B95-8 genome had been extensively mapped for transcripts, promoters, open reading frames, and other structural elements by means of Northern blotting and other methods (13,14). A more representative type 1 EBV reference genome (GenBank accession number NC_007605) was constructed by using B95-8 as the backbone, while an 11-kb deletion segment was provided by the Raji sequences (15).AG876 was originated from a Ghanaian case of Burkitt's lym- (17). C666-1 is a subclone of C666, an epithelial cell line derived from an NPC xenograft of southern Chinese origin (18).…”

mentioning

confidence: 99%

“…Akata and Mutu are African Burkitt's lymphoma cell lines that are commonly used model cell lines. Their EBV genomes were sequenced by next-generation sequencing and constructed by de novo assembly (17). C666-1 is a subclone of C666, an epithelial cell line derived from an NPC xenograft of southern Chinese origin (18).…”

mentioning

confidence: 99%

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

Kwok

Palser

et al. 2014

J Virol

101

111

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Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimenderived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis. IMPORTANCEThis study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.T he incidence rate of undifferentiated nasopharyngeal carcinoma (NPC) is exceptionally high in the southern part of China, and this type of carcinoma is 100% associated with Epstein-Barr virus (EBV) (1). To investigate the role of EBV genomic variation in the pathogenesis of NPC, EBV strains had been characterized in NPC by genotyping polymorphic markers in the EBER1 and -2, LMP1, BHRF1, BZLF1, and EBNA1 gene loci in tumor samples obtained from China, southern Asia, and northern Africa (2-7). Association of LMP1 deletion variant Asp335 with NPC in Hong Kong was reported (8). Specific EBNA1 (V-val) and LMP1 subtypes (China 1) also showed preferential occurrence in NPC biopsy specimens (9, 10). However, genetic variations in the small subsets of genes investigated were not ...

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“…AG876 was originated from a Ghanaian case of Burkitt's lym- (17). C666-1 is a subclone of C666, an epithelial cell line derived from an NPC xenograft of southern Chinese origin (18).…”

Section: Importancementioning

confidence: 99%

mentioning

confidence: 99%

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

Kwok

Palser

et al. 2014

J Virol

101

111

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“…RNA-seq reads were aligned using indexes containing both the human (hg19 assembly) and the Akata EBV (Akata-NCBI accession number KC207813.1 [8]) genomes. Alignments were performed using both Novoalign version 2.08.02 (Novocraft; -o SAM -r R, default options) and Bowtie version 2 (9) (-library-type fr-firststrand, default options).…”

Section: Methodsmentioning

confidence: 99%

Global Bidirectional Transcription of the Epstein-Barr Virus Genome during Reactivation

O’Grady¹,

Cao²,

Strong³

et al. 2014

J Virol

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Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5= and 3= rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. IMPORTANCE Epstein-Barr virus (EBV)is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole. E pstein-Barr virus (EBV) is a human gammaherpesvirus that is prevalent in all human populations. Although infection is frequently asymptomatic, it has been linked to a number of serious diseases such as Burkitt's lymphoma, nasopharyngeal carcinoma, and posttransplant lymphoproliferative disorder (1). Like other herpesviruses, EBV can exist in both lytic and latent phases, with primary lytic infections often progressing to lifelong latent infections with generally sporadic subsequent episodes o...

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“…Genome indexes are generated from fasta files containing each chromosome of the genome of interest and only need to be generated once. This example creates a genome index containing both the human genome and the EBV genome, using a human reference fasta file downloaded from Ensembl and the EBV reference fasta file chrEBV_Akata_inverted.fa [13] downloaded from https://github.com/flemingtonlab/public/tree/master/annotation (see Notes 6--7).…”

Section: Creating Genome Index Files For Star Alignermentioning

confidence: 99%

Analysis of EBV Transcription Using High-Throughput RNA Sequencing

O’Grady

Baddoo

Flemington

2016

Methods in Molecular Biology

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High--throughput sequencing of RNA is used to analyze the transcriptomes of viruses and cells, providing information about transcript structure and abundance. A wide array of programs and pipelines has been created to manage and interpret the abundance of data generated from high--throughput RNA sequencing experiments. This protocol details the use of free and open--source programs to align RNA--Seq reads to a reference genome, visualize read coverage and splice junctions, estimate transcript abundance and evaluate differential expression of transcripts in different conditions. Particular concerns related to EBV and viral transcriptomics are addressed and access to EBV reference files is provided.

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

Cited by 101 publications

References 29 publications

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

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

Global Bidirectional Transcription of the Epstein-Barr Virus Genome during Reactivation

Analysis of EBV Transcription Using High-Throughput RNA Sequencing

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