Epstein-Barr virus DNA detection in brain tissue sections from donors with progressive multiple sclerosis

Epstein-Barr virus DNA detection in brain tissue sections from donors with progressive multiple sclerosis. L. Benincasa, C. Meloni, B. Rosicarelli, C. Veroni, B. Serafini. Department of Neuroscience, Istituto Superiore di Sanità, Rome, Italy Background Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a complex aetiology, influenced by environmental and genetic factors. Epstein Barr Virus (EBV) infection is the main environmental risk factor triggering MS onset, but its contribution to disease progression remains unclear. Studies on post-mortem brain tissue have identified EBV-infected B cells and EBV-specific CD8 T cells in lesions and inflamed meninges of donors with progressive MS (pMS). EBV transcripts were also detected in inflammatory infiltrates isolated from pMS brain, using laser capture microdissection (LCM) and highly sensitive preamplification droplet digital PCR (ddPCR). This study aims to confirm EBV persistence in advanced disease stages by developing a method to detect and quantify viral DNA in pMS brain tissue. Methods For this study, post-mortem MS brain tissue blocks with substantial B cell infiltration were selected. In a first set of experiments, immune infiltrates were isolated from meninges of 8 pMS cases using LCM. DNA was extracted, preamplified and analyzed using ddPCR for the EBV BAMHI-W repeat region along with the human gene EIFC2 as reference. Targeted preamplification became necessary due to the very low DNA yield from the microdissected samples. In a second set, viral DNA was extracted from whole brain sections cut from tissue blocks of 6 pMS cases and analyzed by ddPCR without preamplification Results In LCM samples, EBV DNA was detected in 4 out of 8 meningeal B cell infiltrates. However, viral load quantification was not feasible due to non-linear amplification of the EBV BAM HI W repeat compared to the human EIF2C gene, likely resulting from the approximately 10-fold repetition of the viral region analyzed. In the whole brain sections, EBV DNA was detected in 3 out of 6 samples, with viral load varying depending on the degree of tissue inflammation (median ratio between EBV copies/human EIFC2 copies: 0.00086, range: 0.00007–0.01). This variability may be associated to the number of immune infiltrates (perivascular cuffs/meningeal infiltrates) and the degree of inflammation within the section analyzed. Conclusion Our results confirm the presence and persistence of EBV in the pMS brain. EBV DNA can be detected in post-mortem brain tissue by analyzing microdissected infiltrates and whole sections. In both approaches, selecting brain tissue samples with a substantial B cell infiltration is crucial. While DNA analysis from whole sections is less refined than LCM, it enables viral load quantification. Further experiments will be conducted to validate these findings on a larger sample size.