High Biocontainment (BSL4/ABSL4) core for replication competent virus work

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Core C – Project Summary/Abstract Core C provides support for high biocontainment work to Research Projects 1 (RP01), RP02, RP03 and Core B. Work is prioritized through Core A. Core personnel bring unique expertise in molecular biology, cell biology and animal disease modeling for Ebola and Marburg viruses. The core maintains virus stocks, makes recombinant viruses, provides cell lysates and RNA from infected cells and ensures they are free of infectious material, evaluates effects of host factor loss or overexpression on infection and performs work with transgenic rodents. These core activities provide needed protein material and an understanding of subcellular structures made after virus infection for RP01, are required for evaluation of virus protein function and genome stem loop structures in controlling transcription for RP02, and provide detailed analysis of infection kinetics and host-virus co-association for RP03. To directly address impact of host factors on virus protein production, virus RNA transcription and the substructures formed by each, we have developed a novel protein-antibody + vRNA-FISH based staining technique that is quantitatively assessed by automated microscopy image analysis. This approach is expected to provide important information for the needs of each project and Core B. Core C also works closely with Core B in production and evaluation of novel antibodies by providing purified virus lysates and performing microscopy evaluation of staining of infected cells. The project distinguishes itself in providing high-level molecular biology ability at BSL4 that will be used to produce recombinant viruses predicted to have altered replication kinetics. Furthermore, use of transgenic animals, provided through Core B, will be used to evaluate roles in controlling disease. In so doing, Core C provides the necessary processes to evaluate host factor roles from cells through to disease that enables identification of potential targets for therapy development.