Project Number: 5410-32000-014-05
Project Type: Nonfunded Cooperative Agreement

Start Date: Apr 15, 2004
End Date: Apr 14, 2009

Objective:
To learn new methods utilized by AIDL in evaluating arbovirus susceptibility in mosquito vectors (e.g. Flavivirus susceptibility in Aedes aegypti) and adapt this technology to species of Culicoides.

Approach:
The first phase of this sabbatical proposal is to learn the molecular tools utilized by AIDL by silencing two targets, mucin and a gene associated with inhibition of apoptosis expected to have an effect on dengue virus replication in Aedes aegypti. Two approaches will be used. The first approach will be to clone these target genes into a new Sindbis virus transducing system developed at AIDL. We, in collaboration with AIDL, have shown previously that Sindbis will replicate in Culicoides cells, however, previous recombinant viruses did not replicate in midgut epithelium. This new Alphavirus Transducing System (ATS) is based on an infectious clone of a Malaysian MRE16 Sindbis strain that replicates in Aedes aegypti midgut cells. The effects of infection with the Sindbis recombinant viruses on subsequent dengue virus challenge infection will be evaluated will be assessed by virus titration and immunohistochemistry. The second approach will be to produce small inhibitory RNA (siRNA) by cloning these genes into a RNA transcription vector and using in vitro transcription to produce double-stranded (ds)RNA. The dsRNA will be then be feed or injected into Aedes aegypti, and the mosquito RNA silencing system will produce siRNA. Mosquitoes will then be challenged by infection with dengue virus. The inhibition of infection will be assessed by virus titration and immunohistochemistry. The second phase of the proposal will be to transfer this technology to ABADRL and adapt these systems to Culicoides. Ultimately, the technology will be transferred to the field to confer resistance to bluetongue viruses on Culicoides spp.