Project description:Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here, we use metagenomic RNA sequencing of about 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses, Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not been previously reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and Drosophila obscura, we identify sequences very closely related to Armadillidium vulgare iridescent virus (Invertebrate iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.
Project description:RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.
Project description:The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific.
Project description:Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.
Project description:Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.
Project description:The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. Here, we report that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection.
2018-01-01 | S-EPMC5963806 | BioStudies
Project description:Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV6) Small RNAs deep sequencing
Project description:<h4>Unlabelled</h4>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.<h4>Importance</h4>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.
Project description:Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia-host interaction.
Project description:RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel.