The goal of this research project is to apply molecular genetic techniques to control arthropod-borne (arbovirus) diseases. Arboviruses remain significant pathogens of humans and animals throughout the world. Conventional control measures, including pesticide application, environmental sanitation, and immunization, have proven to be effective in short term reduction in the incidence of arbovirus diseases, but ineffective for long term reduction or erradication. Indeed, many arboviruses remain significant pathogens or are resurgent in many previously controlled areas. Novel strategies are sorely needed for control of vector-borne diseases. In this proposal, novel molecular techniques for control of arboviruses will be investigated. Transposable genetic elements incorporated into molecular constructs will be used to genetically alter the ability of vector mosquitoes to transmit and to maintain arboviruses. The ability of a P element construct containing the heat shock promoter and the neomycin resistance gene to transform Aedes triseriatus mosquitoes will be characterized. Alternate constructs and techniques for transformation, transposition, and assay for expression will be developed and compared for efficiency with the P element based system. Libraries of the mosquito genome will be screened for the presence of efficient promoters that could be incorporated inot constructs to increase expression and for endogenous transposons that could be used to more effectively introduce constructs into mosquito chromosomes. After optimization of transposition and expression systems, attempts will be made to induce interference to arbovirus infection in mosquitoes. Efficient vectors will be genetically engineered to contain cDNA sequences of the La Crosse (LAC) virus genome. Constructs will be microinjected into Ae. triseriatus ova, the natural vector of LAC virus, and the transformed mosquitoes will be assayed for expression of mRNA by Northern blot analyses and for polypeptide expression by Western blot analyses. In situ hybridization and antigen detection will be used to determine the anatomic sites of construct expression. Mosquitoes exhibiting high level expression will be challenged with LAC virus to determine resistance to infection. Resistant specimens will be challenged with related viruses to determine the phylogenetic range of interference. These studies will undoubtedly provide considerable information about the mechanisms of gene regulation and expression in these important vectors of pathogens.
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