EBV genome analysis in multiple sclerosis shows extensive viral diversity and links to autoimmunity

Abstract Epstein–Barr virus (EBV) is a major risk factor for multiple sclerosis (MS), yet the contribution of specific viral variants remains unclear. Complete EBV genome analysis of wild-type variants has not been performed previously. In a pilot-study, targeted EBV sequencing of B cells did not yield adequate coverages due to excess of human DNA. Thus, we developed an ex vivo 6-day leukocyte culture method to enrich EBV DNA into supernatant by stimulation with tetradecanoyl phorbol acetate. Using this method in 20 MS patients and 20 controls, we obtained near-complete EBV genomes in 9 MS patients (average coverage 97%) and in 4 controls (average 85%). We identified 1 088 single-nucleotide variants (33% missense variants) outside repetitive regions that met stringent quality criteria. Overall, 94% of the variants were present in three or more subjects, thus unlikely generated during the culture and several were exclusive to MS, worth of further investigation. In silico analysis indicated that variants within the EBNA1 cross-reactive region change HLA peptide binding affinity, suggesting altered antigen presentation. These results illustrate a method for enriching EBV genomes and present the first wild-type sequences from MS patients. The observed diversity highlights potential for future studies in EBV-associated diseases and vaccine development.