Pathogens

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Project Description:

Due to their high rates of mutation, large population sizes, and rapid rates of replication, RNA virus populations comprise a "quasispecies", or swarm of mutant genomes.  The diversity of a quasispecies influences viral adaptation, disease severity, and response to antiviral therapies. Identifying the mechanisms that shape quasispecies diversity is necessary to better prevent virus emergence and control virus disease, particularly for arthropod-borne viruses (arboviruses), all of which are RNA viruses and many of which are significant emerging threats to global public health. Previous studies have revealed that arbovirus quasispecies diversity may differ between vertebrate hosts and arthropod vectors. In the most complete study it has been shown that quasispecies diversity of West Nile virus is significantly greater in mosquitoes than in birds, suggesting that the antiviral defenses of mosquitoes select for virus diversification. The recent discovery of RNA interference (RNAi), the targeted destruction of RNA with high homology to a double-stranded RNA trigger, has revealed the first general pathway by which arthropods regulate viral replication. The proposed research will test the hypothesis that the RNAi response of vectors imposes selection for mutations that enable virus populations to escape RNAi, and RNAi thereby drives diversification of viral quasispecies. First, two emerging arboviruses, dengue virus and West Nile virus, will be passaged separately in mosquito cells in which RNAi has been specifically activated through treatment with short hairpin RNA's (shRNA's) complementary to three different regions of the viral genome.  At the end of the passage series, quasispecies diversity will be determined by sequencing 50 separate viral genomes from each replicate to test the prediction that activating RNAi will spur quasispecies diversification.  Second, the two viruses will be passaged in Drosophila cells in which RNAi has been depleted  to test the prediction that suppressing RNAi will relax selection for quasispecies diversity.  Finally, both viruses will be passaged in mutant lines of Drosophila which do not express components of the RNAi pathway to test the hypothesis that the loss of RNAi will lead to a decrease in quasispecies diversity.