The specific goal of these studies is to identify the receptor(s) which dengue virus utilizes when infecting its natural hosts, humans and mosquitoes, with the ultimate goal of generating a viable animal model for the study of dengue virus infection and disease. Several approaches are being used to identify the host receptor for dengue infection. First, we are making a panel of monoclonal antibodies against the mosquito cell line, C6/36. Cell membrane preps have been prepared from C6/36 cells and used to immunize mice. Positive mice are currently being assayed for immune response to the C6/36 antigens. Fusions for monoclonal antibodies will be done with positive mice. Resultant monoclonal suprernatants will be screened for their ability to block or inhibit virus binding and or infectivity. Studies are underway to determine the best means for identifying a blocking monoclonal antibody. A second approach to identify the dengue virus receptor in mosquito cells is to make a cDNA libratry from C6/36 cells, express the library in a cell line resistant to dengue infection and assay for positive cells. We have made an expression library from C6/36 cells and have inserted the resultant clones into the pHOOK expression vector system (Invitrogen) which allows for the selection of trasfected cells based on surface expression of the single-chain antibody. We are currently establishing conditions for selecting transfected cells and assaying for expression.
