From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes

Kwok, Harry; Chiang, Alan Kwok Shing

doi:10.3390/v8030060

Cited by 17 publications

(16 citation statements)

References 49 publications

(54 reference statements)

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“…Enrichment of EBV genomes facilitates sequencing directly from patient samples. As noted in previous reports, the small proportion of viral genomes present in even purified B cell fractions presents a challenge for generating complete full-length EBV next-generation sequencing libraries from infected patient samples (29). In the absence of any purification or enrichment strategy, the majority of the sequencing reads align to the human genome.…”

Section: Resultsmentioning

confidence: 99%

“…Next-generation sequencing, coupled with methods used to enrich viral genomes from contaminating human genomes, has proven to be a powerful tool for sequencing and characterization of full-length viral genomes, including EBV. In the past 5 years alone, the number of publicly available EBV genome sequences has increased from under 10 to greater than 100 (26,29). While these studies have provided novel sequence data that increase our understanding of EBV biology, the overwhelming majority of genome sequences originated in either diseased tissue or from immortalized cell lines.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

Weiss

Lamers²,

Henderson

et al. 2018

J Virol

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Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; = -0.5589, = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

Weiss

Lamers²,

Henderson

et al. 2018

J Virol

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“…The tumorigenic propensity of type 1 and type 2 in human cancers is unclear [139]. Recent next-generation sequencing (NGS) examination of EBV has revealed the homogeneous clustering of EBV strains in NPC patients [140]. The specific allelotype of HLA in populations in different geographical regions and the differential ability to present epitopes of different EBV strains may be an underlying selection mechanism.…”

Section: Strain Variations Of Ebv and Npcmentioning

confidence: 99%

Epstein–Barr virus infection and nasopharyngeal carcinoma

Tsao

Tsang

2017

Phil. Trans. R. Soc. B

448

393

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Epstein-Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways.This article is part of the themed issue 'Human oncogenic viruses'.

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“…In positive selection methods, samples are enriched for viral nucleic acids directly using probes targeting the viruses like in PCR assays, microarray or virus capture (in solution based hybridization) approaches. The simplest examples of the positive selection approach are generic PCR assays which use degenerated primers to target several related viruses or their variants ( Irving et al, 2014 , Kwok and Chiang, 2016 , Simons et al, 1995a ). The PCR based approach is restricted by its ability to detect only a limited number of viruses due to an issue of multiplexity.…”

Section: Selective Sequencing Approachesmentioning

confidence: 99%

Evolution of selective-sequencing approaches for virus discovery and virome analysis

2017

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Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once, mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for study of viromes, sample preparation methodologies used different refinements to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focused on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density centrifugation. However, recently an approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was shown to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we have listed the different methods, their applications and evolution, for selective sequencing based virome analysis and also the major refinements needed to harness the full potential of HTS for virome analysis.

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From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes

Cited by 17 publications

References 49 publications

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

Epstein–Barr virus infection and nasopharyngeal carcinoma

Evolution of selective-sequencing approaches for virus discovery and virome analysis

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