1a.Objectives (from AD-416):
To identify new molecular targets to inhibit infection and/or transmission of arboviruses by Culicoides using RNA interference (RNAi).
1b.Approach (from AD-416):
Both ARS and Kansas State University (KSU) have been utilizing molecular tools to investigate mechanism of pathogens infecting insects that transmit them. The RNA interference (RNAi) system will be used for initial evaluation of novel infection blocking targets. Briefly, the 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 long double-stranded (ds)RNA. The long dsRNA will be then be fed or injected into the insect vector, to initiate the insect’s gene silencing system. Biting midges will then be challenged by infection with a single serotype of Bluetongue virus BTV. Gene knockdown will be monitored by qRT-PCR and Western blot where antibodies are available. The inhibition of infection will be accessed by virus titration, qRT-PCR and immunohistochemistry. Preliminary studies have already identified potential inhibition targets for further investigation. The first phase will be to determine if the RNAi technology can be applied to Culicoides on a limited number of target molecules on one serotype of BTV.
The objective of this project was to characterize molecular factors that affect competence of insects for virus infection and transmission with an emphasis on biting midges of the Genus Culicoides. Accomplished during this period was 1. The injection protocol for adult Culicoides sonorensis. 2. The Spn27A ortholog in C. sonorensis as a positive control for RNAi. 3. Production of dsRNAs targeting putative anti-viral immune genes.