Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1-and, by extension, EBV-diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection.

UNLABELLED: Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.).

Date palm provides both staple food and gardening for the Middle East and North African countries for thousands of years. Its fruits have diversified significantly, such as nutritional content, size, length, weight color, and ripping process. Dates palm represent an excellent model system for the study of fruit development and diversity of fruit-bearing palm species that produce the most versatile fruit types as compared to other plant families. Using Roche/454 GS FLX instrument, we acquired 7.6 million sequence tags from seven fruiting stages (F1-F7). Over 99% of the raw reads are assembled, and the numbers of isotigs (equivalent to transcription units or unigenes) range from 30,684 to 40,378 during different fruiting stages. We annotated isotigs using BLASTX and BLASTN, and mapped 74% of the isotigs to known functional sequences or genes. Based on gene ontology categorization and pathway analysis, we have identified 10 core cell division genes, 18 ripening related genes, and 7 starch metabolic enzymes, which are involved as nutrition storage and sugar/starch metabolisms. We noticed that many metabolic pathways vary significantly during fruit development, and carbohydrate metabolism (especially sugar synthesis) is particularly prominent during fruit ripening. Transcriptomics study on various fruiting stages of date palm shows complicated metabolic activities during fruit development, ripening, synthesis and accumulation of starch enzymes and other related sugars. Most Genes are highly expressed in early stages of development, while late developmental stages are critical for fruit ripening including most of the metabolism associated ones.

Large-scale collection and annotation of gene models for date palm (Phoenix dactylifera, L.).

The date palm (Phoenix dactylifera L.), famed for its sugar-rich fruits (dates) and cultivated by humans since 4,000 B.C., is an economically important crop in the Middle East, Northern Africa, and increasingly other places where climates are suitable. Despite a long history of human cultivation, the understanding of P. dactylifera genetics and molecular biology are rather limited, hindered by lack of basic data in high quality from genomics and transcriptomics. Here we report a large-scale effort in generating gene models (assembled expressed sequence tags or ESTs and mapped to a genome assembly) for P. dactylifera, using the long-read pyrosequencing platform (Roche/454 GS FLX Titanium) in high coverage. We built fourteen cDNA libraries from different P. dactylifera tissues (cultivar Khalas) and acquired 15,778,993 raw sequencing reads-about one million sequencing reads per library-and the pooled sequences were assembled into 67,651 non-redundant contigs and 301,978 singletons. We annotated 52,725 contigs based on the plant databases and 45 contigs based on functional domains referencing to the Pfam database. From the annotated contigs, we assigned GO (Gene Ontology) terms to 36,086 contigs and KEGG pathways to 7,032 contigs. Our comparative analysis showed that 70.6 % (47,930), 69.4 % (47,089), 68.4 % (46,441), and 69.3 % (47,048) of the P. dactylifera gene models are shared with rice, sorghum, Arabidopsis, and grapevine, respectively. We also assigned our gene models into house-keeping and tissue-specific genes based on their tissue specificity.

Genome sequence of the date palm Phoenix dactylifera L.

Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm's unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.