UC Riverside

The development of efficient techniques to interrupt the transmission of human pathogenic arboviruses such as Dengue, Zika and West Nile virus is of vital importance as current mosquito control strategies are not sufficient. Insect protection from viruses is typically carried out by the siRNA pathway which involves the degradation of foreign genetic material during an active infection. Recently, another class of small RNAs that form the piRNA pathway have been implicated in antiviral activity in Aedes aegypti. This is unusual as the piRNA pathway canonically functions to protect the germline from the effects of transposons, and hence tends to be limited to gonadal tissues. However, Ae. aegypti was discovered to express piRNAs somatically with recent confirmation of the antiviral activity of piRNAs in the ovaries lending credence to this theory. Not much is known about how the associated effectors of this pathway, known as the PIWI class of proteins, participate in this antiviral activity. Using CRISPR-Cas9 each somatically expressed Piwi gene (Ago3, Piwi4, Piwi5, Piwi6), as well as Piwi2 was knocked out one at a time and the phenotypes of the resulting mosquitoes was characterized. Knockout of each PIWI protein led to a general reduction in viability and fertility of the G0 mosquitoes compared to the control. This suggests that the piRNA pathway may be involved in critical aspects of the development of the mosquito, though further work in vivo is necessary to tease apart the myriad potential functions. The prevalence of endogenous viral elements (EVEs) from which piRNAs arise, in the Ae. aegypti genome, prompted the attempt to generate transgenic mosquitoes primed with immunity to an arbovirus of medical importance. This was done by locating and targeting unique loci in a highly repetitive piRNA cluster that is highly expressed in the midgut, to knock in a segment of the Sindbis virus, after which the transgenic mosquito would test for production of SINV-piRNAs. Establishment of the transgenic line using CRISPaint proved to be challenging, though successful integration was observed. Further investigation of this pathway could result in valuable information on new targets for genetic control of this vector species.