The proposed project will further develop marketable ELISA and lateral-flow immunoassay diagnostics (LFI) in both monoplexed and multiplexed formats for the NIAID category A, BSL-4 filoviruses, and will drive pre-clinical development and validation for these diagnostics. These assays will be used for diagnosis of Ebola virus (EBOV), Sudan virus (SUDV) and Marburg virus (MARV), and will be based on more inexpensive recombinant proteins, rather than reagents that must be produced in high-containment laboratories. We will first scale up production of our existing recombinant filovirus proteins and polyclonal and monoclonal antibodies, in order to meet manufacturing and commercialization requirements. We will next develop second-generation ELISA and LFI prototypes for our existing EBOV, SUDV and MARV antigen detection assays. Third, we will extend the library of available filovirus assays by developing antigen detection tools for the Reston and Bundibugyo viruses. Fourth, we will prepare for commercialization of the assays in both monoplexed multiplexed formats. A multiplexed panel may be ideal for diagnosis as hemorrhagic fevers that are caused by different viruses can present similar symptoms. Antigens from the arenavirus Lassa (LASV), the viral hemorrhagic fever (VHF) most frequently transported out of Africa have already been developed into ELISA and LFI formats by this consortium. Thus, a fifth goal to be accomplished in this project time period is to multiplex LASV antigens with those of the filoviruses in order to develop a broader-scope diagnostic. The resulting multiplexed assays will have utility and marketability for recent travelers who have fallen ill, personnel working in BSL-4 laboratories, in bioterrorism scenarios, and in clinical settings in Africa where these fevers occu naturally. The ultimate goal is to establish a panel of recombinant VHF diagnostics that are easy to use, quick, accurate and cost effective for biodefense and public health purposes. Such diagnostics can be stockpiled to protect civilian and military personnel from viral exposure and release. Viral hemorrhagic fevers are extremely rare in the U.S., but accidental or intentional release could have devastating consequences. Importantly, the simplicity and cost-effectiveness of diagnostics such as these will facilitate the necessary availability of these reagents so that they can be put to immediate use in event of virus exposure or release. Widespread availability of these simple-to-use tests will allow rapid containment of the viruses, and may even serve as a deterrent from using these pathogens as biological weapons.