A single unidirectional piRNA cluster similar to the flamenco locus is the major source of EVE-derived transcription and small RNAs in Aedes aegypti mosquitoes.

Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity.

UNLABELLED: Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCE: Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens.

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host.

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.

dsRNA sensing during viral infection: lessons from plants, worms, insects, and mammals.

Host defense systems often rely on direct and indirect pattern recognition to sense the presence of invading pathogens. Patterns can be molecules directly produced by the pathogen or indirectly generated by changes in host parameters as a consequence of infection. Viruses are intracellular pathogens that hijack the cellular machinery to synthesize their own molecules making direct recognition of viral molecules a great challenge. Antiviral systems in prokaryotes and eukaryotes commonly exploit aberrant nucleic acid sensing to recognize virus infection as host and viral nucleic acid metabolism can greatly differ. Indeed, the generation of dsRNA is often associated with viral infection. In this review, we discuss current knowledge on the mechanisms of viral dsRNA sensing utilized by 2 important antiviral defense systems, RNA interference (RNAi) and the vertebrate immune system. The major viral sensors of the vertebrate immune systems are RIG-like receptors, while RNAi pathways depend on Dicer proteins. These 2 families of sensors share a similar helicase domain with high specificity for dsRNA, which is necessary, but not sufficient for efficient recognition by these receptors. Additional intrinsic features to the dsRNA molecule are also necessary for activation of antiviral systems. Studies utilizing synthetic ligands, in vitro biochemistry and reporter systems have greatly helped increase our knowledge on intrinsic features of dsRNA recognition. However, characteristics such as subcellular localization are extrinsic to the dsRNA itself, but certainly influence the recognition in vivo. Thus, mechanisms of viral dsRNA recognition must address how cellular sensors are recruited to nucleic acids or vice versa. Accessory proteins are likely important for in vivo recognition of extrinsic features of viral RNA, but have mostly remained undiscovered due to the limitations of previous strategies. Hence, the identification of novel components of antiviral systems must take into account the complexities involved in viral recognition in vivo.