Characterization of Epstein-Barr virus genotypes in multiple sclerosis

Objective: Converging epidemiological, clinical and laboratory studies support an etiologic role for Epstein Barr virus (EBV) in MS. Nonetheless, it is difficult to understand how a virus that ubiquitously infects humans can be associated with a disease of relative limited prevalence. The possibility that only a particular viral genotype associates with the disease has been proposed and investigated by many groups, with conflicting results. With the aim to shed light on this controversial scenery we carried out a study aimed to the characterization of Epstein-Barr virus genotypes in MS patients. Methods: Genomic DNA was extracted from CD19+ B cells or peripheral blood mononuclear cells (PBMCs). All samples were analyzed by nested PCR approach using EBNA2 type specific primers and PCR products were assayed by a standard sequence analysis. We addressed to high-throughput sequencing by the Roche 454 platform the PCR amplicons obtained from two large genomic regions of OriP and EBNA1. Results: We characterized 39 MS patients and 33 HD for EBNA2 genotypes in a region of about 500 bp length. A significant bias in the distribution of EBV subtypes was observed. In 4 MS and 4 HD we performed experiments in which we attempted to sequence longer stretches (about 1000 bp) of the viral genome within the OriP and EBNA1 regions. The shotgun sequencing of the two amplicons generated a total of about 800000 reads of high quality, most of them having a length consistent with the expected one of about 400 bases. Conclusions: We detected several variants with respect to the known EBV genomes and will present data on their differential representation in MS and normal samples