1a.Objectives (from AD-416):
To identify new molecular targets to inhibit infection and transmission of arboviruses by mosquito vectors of these animal and zoonotic pathogens using RNA interference (RNAi).
1b.Approach (from AD-416):
Utilizing Malaysian MRE16 Sindbis strain that replicates in Aedes aegypti midgut as a model arbovirus to assess molecular inhibition targets, both parties have identified potential but distinct inhibition targets for further investigation. This model system will be further used for initial evaluation of additional novel inhibition targets that have been identified in another recently describe model system. The inhibition 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 the model virus. The inhibition of infection will be assessed by virus titration, qRT-PCR and immunohistochemistry. The second phase of the proposal will be to evaluate proven targets ability to inhibit Rift Valley fever virus infection of and transmission by mosquitoes once appropriate facilities are available.
The Objective of this agreement is to develop novel molecular targets that disrupt infection and/or transmission of virus by the insects that transmit them. During the past year, effort has been focused on the Dnr1 gene from Aedes aegypti. Evidence obtained from Drosophila and A. aegypti suggests that Dnr1 is involved in regulating apoptosis and immunity. Experiments have been initiated that are designed to silence the expression of Dnr1 in adult Ae. aegypti mosquitoes using RNA interference. Once the gene silencing has been optimized, the effect of decreased Dnr1 levels on arbovirus infection will be studied. The level of expression of Dnr1 during infection of Ae. aegypti by arboviruses is also being examined using quantitative PCR.