2012 Annual Report 1a.Objectives (from AD-416): To indentify 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): Approach: Both ARS and KSU have been utilizing Malaysian MRE16 Sindbis strain that replicates in Aedes aegypti midgut as a model arbovirus to assess molecular inhibition targets. This system will be used for initial evaluation of novel inhibition targets that will be assessed by virus titration and immunohistochemistry. 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 and immunohistochemistry. Preliminary studies at KSU and ARS have already identified potential inhibition targets for further investigation. A 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. Future studies may include the adaptation these systems to Culicoides. 3.Progress Report: 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. Initial progress with previously designed target led to the development of funded Specific Cooperative Agreement #58-5430-1-0358 with Kansas State University.