Program Summary Progressive Multifocal Leukoencephalopathy (PML) is a major life threatening complication in patients with AIDS and in patients undergoing immunotherapy for autoimmune diseases such as multiple sclerosis, Crohn's disease, severe plaque psoriasis, systemic lupus erythematosis, hematologic malignancies, and rheumatoid arthritis. The disease is paradoxically caused by a common human polyomavirus following the loss of normal immune surveillance of the central nervous system (CNS). There are several major gaps in our understanding of the basic biology that underpins the development of PML. First, the anatomical site of virus persistence is not known but kidney, bone marrow, and brain have all been postulated to be involved. Second, the mechanisms that govern viral persistence are not well defined but changes in the viral promoter (archetype to PML-type) and regulation of a viral microRNA are thought to be critical for transition to the lytic phase. Third, the mechanisms of viral spread to the CNS and within the CNS are not known. Based on our recent work we hypothesize that free virus as well as virus enclosed in extracellular vesicles spreads from the kidney to the choroid plexus and meningeal compartments where replication in CPE cells provides the means for EV mediated invasion of brain parenchyma. Once in the brain extracellular vesicle mediated spread is likely to play the major role in spreading the infection because macroglia lack the principle cellular receptors to support infection by free virus. Our vision for the R35 application is to further our understanding of these basic mechanisms of infectious spread of virus and to use the information to identify biomarkers that can predict PML early and in easily accessible compartments well before the virus has a chance to spread to the CNS. The work should also lead to the development of novel strategies to treat and prevent PML.