Genome Diversity of Epstein-Barr Virus from Multiple Tumor Types and Normal Infection

Abstract

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 Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis

“…The EBV genome encodes fewer proteins than the vaccinia virus genome but many more than the genome of HIV or influenza virus, providing ample structural proteins to serve as CD4 T-cell epitopes. In agreement with our findings, previous analyses of available full-length EBV genomes, although mostly from cell lines and tumors, have shown the gp350 gene to be one of the least variable genes, suggesting that our measurements of gp350 gene variation may significantly underrepresent the total viral genome variation (18).…”

“…Recent studies, including our own, have demonstrated that dsDNA viruses can exhibit levels of variation similar to those observed in some RNA viruses, such as West Nile virus and HIV-1 (16,17). Most published EBV gp350 sequences have originated from either transformed B-cell lines or cancerous tissue; at present, only a very limited number of primary EBV gp350 sequences are available (18). A better understanding of the diversity of circulating EBV gp350 sequences, as well as the potential role of EBV-specific antibodies in the evolution of EBV gp350 diversity over time in infected individuals, would be helpful to inform vaccine development.…”

Tumor microenvironment contributes to Epstein-Barr virus anti-nuclear antigen-1 antibody production in nasopharyngeal carcinoma

“…EBNA1 is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies (9,10). It is potentially a universal target for immune recognition of EBV-infected normal or malignant cells (10).…”

“…EBNA1 is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies (9,10). It is potentially a universal target for immune recognition of EBV-infected normal or malignant cells (10). The series of essential components for a humoral immune response, including NA1-expressed cells, APCs (HLA-DR+, CD80+ and CD86+), T helper cells (CD3+ and CD4+) and B cells (CD19+ and IgA+), were all detected by IHC staining in the local tumor environment.…”

“…Nuclear antigen-1 (NA1) protein is expressed during latent and lytic cycles, binding to a replication origin within the viral genome. It also mediates replication and partitioning of the episome during the division of the host cell, and is essential for the maintenance of the viral genome in latency (8)(9)(10)(11). Since NA1 protein is expressed in EBV-infected cells, including cancer cells or cells in the cancer microenvironment, it may act as an antigen to induce immune responses if the microenvironment is suitable for the induction of humoral responses directed at NA1 (7).…”