RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Here, we show that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms.

Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. Here, we demonstrate that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. Our work also describes a molecular framework for the viral immunity, in which viral doublestranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals.

In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.arboviruses | piRNAs | siRNAs | viral immunity | massively parallel sequencing T he Dicer family of host immune receptors mediates antiviral immunity in fungi, plants, and invertebrate animals by RNA interference (RNAi) or RNA silencing (1-3). In this immunity, a viral dsRNA is recognized by Dicer and diced into siRNAs. These virus-derived siRNAs are then loaded into an RNA silencing complex to act as specificity determinants and to guide slicing of the target viral RNAs by an Argonaute protein (AGO) present in the complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain shared with AGOs, and two tandem type III endoribonuclease (RNase III) domains. Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA, producing duplex small RNA fragments of discrete sizes progressively from the terminus (4).In addition to siRNAs, micro-RNAs (miRNAs) and PIWIinteracting RNAs (piRNAs) guide RNA silencing in similar complexes but with distinct AGOs (4-6). In Drosophila melanogaster, miRNAs and siRNAs are predominantly 22 and 21 nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis, and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily, respectively (4-6). In contrast, ∼24-30-nt piRNAs are Dicer-independent and require AGO3, Aubergine (AUB), and PIWI in the PIWI subfamily for their biogenesis (4-6). Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2 in the immunity and biogenesis of viral siRNAs targeting diverse positive-strand (+) RNA viruses, including Flock house virus (FHV), cricket par...