SWINE INFLUENZA VIRUSES CAUSE ACUTE RESPIRATORY INFECTION IN SWINE HERDS AND SIGNIFICANT ECONOMIC AND ANIMAL LOSSES TO THE SWINE INDUSTRY. IN THE UNITED STATES ALONE, THE PORK INDUSTRY LOST AN ESTIMATED $5 BILLION DUE TO THE 2009 H1N1 SWINE FLU OUTBREAK. SWINE CAN BE INFECTED WITH FOUR DIFFERENT INFLUENZA TYPES THAT VARY IN THE SEVERITY OF INFECTION. OF THE FOUR INFLUENZA VIRUSES, INFLUENZA A IS THE MOST PREVALENT, AND CAUSES THE HIGHEST MORBIDITY AND MORTALITY IN SWINE. EFFECTIVE VACCINES AGAINST INFLUENZA VIRUSES HAVE BEEN DEVELOPED AND ARE CURRENTLY THE BEST PREVENTATIVE TREATMENT OPTION AVAILABLE. HOWEVER, THESE VACCINES ARE NOT EFFECTIVE IN TREATING ALREADY INFECTED ANIMALS. IN ADDITION, NEW SWINE INFLUENZA VARIANTS ARISE THAT ARE NOT TARGETED BY VACCINES, AND THESE VIRUSES PERIODICALLY SPILL-OVER TO OTHER HOSTS INCLUDING HUMANS.INFLUENZA VIRUSES CANNOT REPLICATE ON THEIR OWN AND TAKE ADVANTAGE OF SWINE PROTEINS TO INFECT, REPLICATE, AND SPREAD TO OTHER CELLS. AS SUCH, INFLUENZA VIRUSES ARE ABSOLUTELY DEPENDENT ON HOST CELLS TO PROPAGATE AND SPREAD. TO LIMIT VIRAL REPLICATION AND SPREAD, SWINE CELLS USE A LIMITED DEFENSE SYSTEM AGAINST VIRAL PATHOGENS SUCH AS INFLUENZA. SWINE CELLS EXPRESS PROTEINS THAT LIMIT OR CREATE UNFAVORABLE CONDITIONS FOR VIRAL REPLICATION. NEW ANTI-INFLUENZA THERAPEUTIC STRATEGIES COULD POTENTIALLY BE DEVELOPED IF WE IDENTIFY ALL INFLUENZA DEPENDENCY FACTORS OR SWINE DEFENSE PROTEINS.TOWARDS THIS GOAL, WE WILL USE EMERGING GENE-EDIT TECHNOLOGIES TO DELETE EVERY SWINE GENE IN POOLS OF CELLS, WHERE ONE CELL HAS EXACTLY ONE DELETED GENE AND THE NEIGHBORING CELL HAS A DIFFERENT DELETED GENE. POOLS OF GENE-EDITED CELLS WILL BE INFECTED WITH A FLUORESCENT INFLUENZA VIRUS TO VISUALIZE INFLUENZA INFECTION IN CELLS. SWINE CELLS MISSING PROTEINS REQUIRED FOR INFLUENZA REPLICATION ARE EXPECTED TO PRODUCE LOW TO UNDETECTABLE INFLUENZA VIRUS LEVELS AS JUDGED BY LOW TO NO FLUORESCENCE. ON THE OTHER HAND, SWINE CELLS MISSING PROTEINS THAT FUNCTION TOLIMIT VIRAL REPLICATION ARE EXPECTED TO HAVE HIGH LEVELS OF INFLUENZA VIRUS AS JUDGED BY HIGHER FLUORESCENCE WHEN COMPARED TO NORMAL UNEDITED SWINE CELLS. NEXT-GENERATION DNA SEQUENCING METHODS WILL BE USED TO IDENTIFY DELETED GENES IN LOW AND HIGH FLUORESCENT CELLS, AND THE TOP GENES IDENTIFIED WILL BE VALIDATED AND SUBSEQUENTLY CHARACTERIZED. THIS STUDY WILL NOT REQUIRE THE USE OF LIVE ANIMALS AND WILL INSTEAD UTILIZE CELLS DERIVED FROM RESPIRATORY TISSUES SUCH AS SWINE TRACHEA AND LUNGS.OUR APPROACH IS UNCONVENTIONAL IN THAT WE WILL TARGET THE HOST CELLS RATHER THAN THE VIRUS ITSELF. WHILE INFLUENZA VIRUSES HAVE THE ABILITY TO CAUSE ILLNESS, THEY ALSO HAVE A CRITICAL WEAKNESS - THEY ARE UNABLE TO REPRODUCE ON THEIR OWN AND DEPEND ON THE PROTEIN-MAKING MACHINERY OF THEIR HOST TO PROPAGATE. THIS WEAKNESS IS WHAT WE ENVISION EXPLOITING AS AN ALTERNATIVE APPROACH TO COMBAT INFLUENZA VIRUS. THIS STUDY WILL CREATE NEW KNOWLEDGE AND TOOLS TO FACILITATE THE DEVELOPMENT OF NEW ANTI-INFLUENZA THERAPEUTICS THAT MIGHT FUNCTION TO BLOCK PROTEINS REQUIRED BY INFLUENZA VIRUSES AND/OR ACTIVATE SWINE ANTI-VIRAL DEFENSE SYSTEMS.