A critical barrier for the treatment of viral encephalitis is the ability to effectively treat patients after they present to their physicians when the virus as likely infected neuronal cells. Unfortunately, neurons are typically not neuro-regenerative, so therapeutic modalities should focus on preventing the death of neurons, as well as to limit virus replication with compounds that can effectively penetrate the blood brain barrier. This project will develop a strategy to accomplish this. The purpose of the R21 phase of the project is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)-induced death, and to establish the utility of specific cellular metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on cell culture-efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents, and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). The specific aims are based on published results that WNV infection in different cell lines leads to Ca++ influx, which benefits is replication, but induces caspase 3 cleavage and increase the risk of cell death. Conversely, inhibitors of Ca++ influx decreases viral titer by >2 log10, and decreased caspase 3 cleavage. In an innovative hamster model of WN neurological disease, induction of calbindin D28k correlates with neuronal survival. Conversely, low basal levels of calbindin D28k correlates with increased TUNEL staining, and cell injury or death. Moreover, the specific metabolites to be investigated are well recognized to co-translocate to the nucleus with the receptor to activate production of calcium buffer proteins. The Specific Aims are: R21 SA #1. Test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV-induced death. R21 SA #2. Establish a neuro- protection cell culture assay for evaluation of vitamin D3 metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. R33 SA #1. Identify lead candidate of vitamin D3 metabolites using the neuro-protective cell culture assay and a transendothelial permeability assay. R33 SA #2. Obtain preclinical data supporting the treatment of WNV encephalitis with lead candidate in animals with different routes of administration and treatment schedules. R33 SA #3. Determine the broad-spectrum therapeutic activity in JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro- pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general. PUBLIC HEALTH RELEVANCE: The purpose of the R21 phase is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)- induced death and to determine the utility of specific metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents for West Nile virus (WNV), and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). To test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV- induced death, we will correlation of neuronal survival with induction of calbindin D28k in the hamster model. We will also test if increased levels of calcium binding proteins from expression vectors will increase the survival of WNV-infected cells and rodents. We will establish a neuro-protection cell culture assay for evaluation of selective metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. Candidates will also be screened using a BBB transendothelial permeability assay. These assays will be necessary to select the most efficacious metabolite. The lead test agent will be evaluated in hamsters and in knockout transgenic mice. We will obtain preclinical data on more appropriate parenteral or oral treatment regimens in animal models. If there is evidence for animal efficacy of the lead metabolite, there is a possibility that dietary deficiency could correlate with severe neurological disease. Therefore, studies with deficient, normal and super levels of the metabolite in the diet of hamsters will be conducted. Also, serum samples from a cohort of 100 WNV patients will be assayed for the surrogate metabolite. In light of similarities of JEV with WNV, and that JEV is listed in the RFA instructions, the therapeutic calcium binding target will also be investigated fo JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover a therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro-pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general.