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 arboviruses by the insects that transmit them. Efforts during the past year have mainly continued to focus on the dnr1 gene from Aedes aegypti. Evidence obtained from Drosophila and A. aegypti suggests that Dnr1 is involved in regulating apoptosis and immunity. Previous work showed that silencing expression of Aedes dnr1 by RNA interference causes spontaneous apoptosis in cultured Ae. aegypti cells. Expression of Aedes dnr1 was silenced in adult female Ae. aegypti mosquitoes by RNA interference. Although dnr1 silencing was only marginally effective as determined by quantitative RT-PCR, it resulted in increased mosquito mortality, which correlates with its phenotype in cultured cells. Expression of dnr1 during arbovirus infection is currently being evaluated. A polyclonal antiserum was raised against Dnr1, and this antiserum is currently being evaluated for use in immunoblotting and immunofluorescence assays. In addition to experiments on Dnr1, we also began cloning full length cDNAs corresponding to four Ae. aegypti caspases, which have not been studied. The potential involvement of these caspase genes in arbovirus infection will be evaluated.