BACKGROUND: Mosquitoes and other blood-feeding arthropods transmit important human and animal viruses (arthropod-borne viruses, arboviruses). With the increasing global threat of arboviruses, it is essential to understand the virus-vector interactions that determine virus transmission. The mosquito antiviral immune response is a key determinant of virus replication and transmission. We recently showed that arboviruses are targeted by a poorly-understood RNA silencing pathway in the major vector mosquito Aedes aegypti: the Piwi-interacting RNA (piRNA) pathway. Our (published and unpublished) observations imply that the piRNA pathway contributes to antiviral defense against different classes of viruses in somatic tissues of mosquitoes. Moreover, we identified a novel class of endogenous gene-derived piRNAs in mosquitoes that may form a new paradigm for piRNA-based regulation of cellular gene expression.AIM: This proposal has a three-fold aim: i) to delineate the biogenesis and function of the novel classes of virus- and gene-derived piRNAs, ii) to characterize mechanisms by which (arbo)viruses suppress or evade antiviral RNA silencing pathways, and by doing so, iii) to establish mosquitoes as an experimental model to characterize the complex piRNA machinery.APPROACH: We will use Aedes cell lines that recapitulate all aspects of piRNA biogenesis. This allows us to use a unique, powerful approach of genomic, cell biological, biochemical, and proteomic methodologies to study piRNA biogenesis and function.IMPORTANCE AND INNOVATION: This is the first study to comprehensively characterize viral and cellular piRNA biogenesis and function in mosquitoes. This proposal provides novel insights into the antiviral response in mosquitoes and may uncover novel regulatory functions of endogenous piRNAs. Moreover, it establishes a platform for functional and biochemical dissection of the complex biogenesis of piRNAs - the most enigmatic class of small silencing RNAs.