PARTNERSHIP: FUNCTIONAL GENOMICS APPROACH IN LIVESTOCK TO DELINEATE HOST FACTORS CRITICAL FOR EMERGING VIRUS REPLICATION

<b>Project Methods</b><br> We intend to make immortalized cell lines from avian intestinal and lung epithelial cells. Necropsied chickens will have GI tract and lungs used to make single cellular suspensions. Lentivirus will be used to immortalize the cells with chicken htert and telomerase. Cell lines including human intestinal epithelial, swine intestinal epithelial, and chicken intestinal epithelial will be infected with PDcoV. RNA will be harvested and RNAseq will be utilized to visualize to the host resposnes to viral infection. Genomic CRISPR knockout libraries are created and maintained by Fun Grey and J Kenneth Baillie at the Roslin Institute and the human library can be purchased commercially from Addgene. Libraries will be created in cognate cell lines swine testiculur, IPEC-J2 (pig), BJ and HIEC human), and avian intestinal epithelium and DF-1 and (chicken) cell lines. Next generation sequencing will be used to ensure the library covers all of the representative genes. Once confirmed, these cell lines will be infected with a high titer virus (PEDV, TGEV, PDCoV). Surviving cells (Resistant to virus infection) will be sequenced to determine which host factors were absent in those cells. Confirmed resistance genes will be verified by making cell lines with specific genes removed and resistance to infection will be confirmed.