application abstract): The immediate objective of the proposed study is to create a recombinant dengue virus containing a suitable reporter gene which will allow direct analysis of viral infection in living cells. To clone a reporter gene, cDNA copy of the dengue virus type 2 will be constructed which will be used to produce full length infectious viral RNA. The potential sites for insertion of reporter gene sequence will be analyzed first, the genetic stability of insertions, the replication competence in terms of rate of virus growth and plaque size. Insertion sites that possess the greatest stability and cause minimum change in phenotype will be selected. Reporter genes such as those coding for green fluorescent protein GFP will be first tested. The expression of the reporter gene in cultured cells and living mosquitoes will be detected. The long range objective of this work is to use tagged virus to enhance our understanding of virus infection and transmission with respect to the viral factors that control cellular infection and pathogenecity, rapid and direct measure of virus replication in cultured cells and transmission in mosquitoes, as well as to develop a functional assay for the identification of the dengue virus receptor on the surface of mammalian cells.
