Development of Aedes aegypti densovirus as a vector for RNA interference

Aedes aegypti mosquitoes are the principal vector species for diseases of significant world health importance including yellow fever and dengue. Aedes aegypti densovirus ( AeDNV) is a parvovirus that infects and kills Aedes aegypti mosquitoes. AeDNV has demonstrated potential as a biological control agent and as a vector for the expression of foreign genes in Aedes aegypti mosquitoes. The hypothesis for these studies was that AeDNV could be developed as a vector for RNA interference (RNAi) in mosquitoes.; cDNA sequence analysis was used to determine that polyadenylation of AeDNV transcripts occurs in the right-end UTR of the virus genome 13 nucleotides downstream of a canonical AATAAA polyadenylation hexamer. Mutational analyses of the AeDNV right-end UTR were used to determine the sequence elements required for efficient gene expression from viral promoters. Efficient gene expression requires the presence of a 21 nucleotide upstream sequence element (USE) that is predicted to form a stem-loop secondary structure in the RNA transcript. Deletion analysis also identified non-essential sequences downstream of the poly(A) site suitable for replacement with an RNAi expression cassette.; Polymerase III (Pol III) promoters capable of mediating RNAi in mosquito cells were cloned from the Anopheles gambiae and Aedes aegypti genomes. The Pol III promoters were tested for the ability to express short-hairpin RNAs (shRNA) targeted to firefly luciferase and to induce RNAi-mediated knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells.; The infectious clone of AeDNV (pUCA) was modified to include a Pol III promoter-based RNAi expression cassette in the right-end UTR of the virus genome. The modified genomes were packaged into AeDNV virions that were capable of infecting and killing Aedes aegypti mosquitoes. Sequence analysis of viral DNA harvested from adult mosquitoes infected as larvae indicated that the inserted RNAi cassettes are stable throughout the duration of the mosquitoes' life span.