Multiple sclerosis (MS), systemic lupus erythematosus (SLE) and type 1 diabetes (T1D) are autoimmune diseases that arise from dysregulation and aberrant activation of B and T cells, which attack the patient’s own tissue. The factors initiating loss of tolerance in many autoimmune diseases are poorly understood and most likely the result of a combination of genetic and environmental factors. Environmental factors can include lack of sun light and vitamin D, cigarette smoke, and infections with certain viruses and bacteria. Epstein-Barr virus (EBV) is thought to be a possible trigger for loss of tolerance in many autoimmune diseases, in particular MS and SLE. Epidemiological studies demonstrated that 99.5% of MS and SLE patients are infected with EBV, often years prior to disease onset. Although the EBV infection rate in the unaffected population is also high (~94%), the difference in EBV infection rate between affected and unaffected populations is significant. Nevertheless, how EBV might promote MS and/or SLE is poorly understood. EBV primarily targets B cells, and the viral genome can survive within B cells for years after initial infection. B cells are known to play important roles in MS and SLE, as demonstrated by the success of B cell targeting therapies in both diseases. We can distinguish EBV-infected from non-infected B cells by single-cell RNAsequencing. Further, studies demonstrated that genomic variants of EBV are responsible for certain tumors, and there is initial data suggesting that EBV variants might be associated with MS. Further, antibody titers against certain EBV proteins are elevated in MS patients, while our preliminary studies that demonstrate that B cells from MS and SLE patients produce anti-EBV antibodies which cross-react to myelin proteins. Together, these findings suggest that EBV might play a key role in mediating MS and/or SLE. Our proposal is to investigate the role of EBV-infected B cells in MS and SLE. We propose to study the influence of EBV on B cells in MS and SLE patients from three different angles: (i) Investigate cross-reactivity of antibodies against EBV and self-tissue, (ii) Identify how EBV activates B cells to promote autoimmunity in MS and SLE, and (iii) Identify EBV strains that are prone to causing MS and/or SLE. We propose to use a unique set of next-generation methods to investigate the role of EBV in MS and SLE. Success of this proposal would identify the molecular mechanisms by which EBV promotes MS and SLE, and could lead to development of vaccines and next-generation therapeutic approaches for MS and SLE.