INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES

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The long term goal of this research is to define molecular approaches for promoting intracellular interference to dengue (DEN) virus infections within Ae. aegypti mosquitoes. Novel expression systems based on infectious clones of mosquito-borne alphaviruses [Sindbis (SIN); Togaviridae] will be used to express either specific DEN-2 viral genes or RNA sequences which can potentially induce interference to virus replication in both mosquito cells and mosquitoes. These approaches will use virus interference strategies which have been successful in generating disease resistant transgenic plants. Such an approach has never before been possible in mosquitoes and its utilization in this work represents an unprecedented opportunity to disrupt the virus-vector-host cycle. Strategies which prove to be promising can eventually be used to generate virus resistant transgenic mosquitoes. The SIN virus expression vectors we propose to use employ a binary system to generate recombinant virus which is infectious for only a single replicative cycle. This is accomplished by cotransfecting susceptible cells with RNAs that have been transcribed (in vitro) from two different plasmids. The RNA from one plasmid consists of the nonstructural genes and internal initiation site for the 26S mRNA of SIN virus. An exogenous gene is inserted downstream of the internal initiation site and replaces the viral structural genes. RNA from the second plasmid consists of a SIN infectious clone in which the NSP1 capsid binding site has been deleted but which encodes the complete structural gene region of the virus. Only the defective RNA from the first plasmid can be packaged to form an infectious virion. This has obvious benefits when considering safety issues related to work with recombinant viruses. We propose to insert DEN virus sequences into this vector in both sense and antisense orientations. Initial studies will be with mosquito cells infected with the recombinant viruses and challenged with DEN-2 virus. If the strategies prove to be successful in causing interference, then equivalent experiments will be performed in mosquitoes. We also plan to use the SIN double-promoter expression plasmids for analysis of interference in mosquitoes. These viral expression vectors generate fully infectious recombinant virions which will allow dissemination of the virus throughout the mosquito. These studies will provide considerable information about the mechanisms of interference at the level of gene expression and in addition, will give information pertinent to current research on the production of transgenic arthropods with reduced vector competence.