West Nile Virus (WNV) has been responsible for an estimated 3 million infections resulting in approximately 780,000 illnesses from 1999 to 2010. Surprisingly, the 2012 WNV season saw a dramatic increase in reported cases compared to the previous five year period, with more than 5,380 reported infections, of which 50% included neuroinvasive disease and 243 resulted in death. After WNV transfusion transmission was demonstrated in 2002, nucleic acid amplification technology (NAT)-based screening of US donors was implemented in 2003. Refined NAT screening strategies and deferral policies have prevented thousands of WNV transfusion transmissions since their implementation. Recently, we demonstrated that while WNV RNA is cleared from the plasma fraction of the blood within 2 to 3 weeks, it can persist for months in whole blood. NAT screening is currently performed on plasma; therefore, blood/organ donors who are WNV-negative by NAT in plasma may still have WNV in other blood compartments and tissues. Considering the lack of reported transfusion-transmission cases in recipients of low-level viremic units, it is generally accepted in the transfusion field that low-levels of WNV persisting after seroconversion are neutralized by antibodies and not infectious. However, studies have shown the possible persistence of infectious particles in different blood compartments and tissues despite the presence of anti-WNV antibodies. Our findings of WNV RNA persistence in whole blood despite clearance in plasma warrants further investigation to determine if there is a residual risk of WNV transfusion or organ transplantation transmission after seroconversion, and if infection can be transmitted to immunocompromised patients, who have increased risk for the development of severe disease outcomes. The research strategy in this proposal was designed to address the remaining risk of WNV transmission in a murine model both in immunocompetent and immunocompromised animals, with the understanding that many transfused and transplanted patients have underlying conditions that render them more susceptible to infectious agents. The findings will inform the need to mitigate any remaining risk of WNV transmission with alternate WNV screening platforms using whole blood instead of plasma, mandatory screening of organ donors for WNV, and careful monitoring of organ recipients for the development of WNV illness and administration of WNV prophylaxis treatment (when available) to recipients of organs collected from donors who test positive for WNV in whole blood or have recently seroconverted. In addition nucleic acid cell-associated persistence, observed during the tail-end of the viremia after WNV and parvovirus B19 infections, may similarly occur after infection with other blood-borne pathogens such as dengue, HCV and HIV. Therefore, using WNV as a model, the present proposal may help to better understand and prevent blood-borne pathogen transmission.