Project Summary / Abstract Central nervous system infections with RNA viruses cause severe neurological deficits and death, and are extremely challenging to diagnose. This multiple PI proposal presents an innovative research program that will provide the foundation for a comprehensive viral diagnostics pipeline to rapidly diagnose RNA virus infections from surgical brain biopsies. The investigators will combine two cutting-edge techniques for viral detection, screening with double-stranded RNA immunohistochemistry (dsRNA IHC) to identify cases with a high likelihood of a viral etiology, and unbiased metagenomic next-generation sequencing (mNGS) for specific virus identification and genome analysis. To develop the dsRNA IHC screening assay, six commercially available anti-dsRNA antibodies will be optimized in formalin-fixed, paraffin-embedded (FFPE) brain tissue and evaluated in a series of known infectious and non-infectious cases. The top candidates will be tested in a set of brain biopsies with inflammation of unclear etiology and compared to results of mNGS performed in parallel. The development of this assay will facilitate efficient screening of FFPE tissue samples from patients with neurological disease of unclear pathogenesis to identify those with a high likelihood of viral etiology. To improve mNGS methods for FFPE brain tissue, new laboratory techniques will be tested to increase the yield and quality of viral RNA extracted. In addition to virus identification, mNGS will be used to perform full-genome deep sequencing of viruses. These results can be applied to molecular epidemiology, disease surveillance, and understanding pathogen-host interactions. Overall, this project will enhance our understanding of RNA viruses that cause CNS infection, inform future studies of virus evolution and pathogenesis, and lay the foundation for the development of a comprehensive viral diagnostics pipeline that will substantially improve the care of patients with devastating CNS infections. This work aligns very well with the NINDS mission to seek and apply fundamental knowledge of the brain to reduce the burden of neurological disease. This R21 Exploratory Neuroscience Research Grant proposal will support and expand the collaboration between the two PIs. The unique combination of expertise in neuropathology, infectious disease pathology, virology, clinical infectious disease, and metagenomic sequencing demonstrate the requisite skills and perspective needed to achieve the goals of this proposal. The stimulating environments of Brigham and Women’s Hospital, Harvard University, and Emory University are extremely well suited to the proposed research plan, which not only provide critical equipment and facilities infrastructure, but also mentorship, formal opportunities to present and discuss results, and eventual venues to implement this clinical testing prospectively.