SUMMARY The central goal of this project is to examine antiviral immunity in insects. Insect transmission of arboviruses causes widespread and debilitating disease across the globe. Viral replication and dissemination in the vector are critical factors in transmission competence. RNA interference is the insect major antiviral system that inhibits viral replication and viral dissemination throughout the insect tissues. The mechanisms involved in developing and effective antiviral immunity are poorly understood. We propose to study antiviral immunity in the model organism Drosophila melanogaster. We discovered that immunity involves uptake of viral double stranded RNA by hemocytes, followed by reverse transcription to generate viral DNAs (vDNAs) that enables amplification of the RNAi response by de novo synthesis of siRNAs (secondary vsRNAs). Central to Drosophila immunity are macrophage-like cells, hemocytes, which are responsible for antiviral RNAi amplification by the production of 5’-triphosphorylated secondary vsRNAs. In the effector face, vsRNAs are incorporated into exosome-like vesicles (ELVs). Haemocyte-derived ELVs mediate the delivery of antiviral vsRNAs to uninfected tissues and protect flies from infection at distal sites. In striking parallel to vertebrates, flies also rely on systemic immunity to control viral infection, albeit in this case the virus-specific signal is nucleic acid-based. We hypothesize that haemocytes take up dsRNA from infected cells to direct the synthesis of vDNA, which in turn templates secondary vsRNA providing adaptive immunity. We will examine (i) the mechanisms of vDNA synthesis, (ii) production and regulation of vsRNA production and (iii) the generation of antiviral ELVs and their function in antiviral immunity.