Virus Recognition by Toll-7 Activates Antiviral Autophagy in Drosophila

SummaryInnate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways

Genome-wide RNAi screen identifies broadly-acting host factors that inhibit arbovirus infection

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts

Stem-Loop Recognition by DDX17 Facilitates miRNA Processing and Antiviral Defense

SummaryDEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements

WSES guidelines for management of Clostridium difficile infection in surgical patients

In the last two decades there have been dramatic changes in the epidemiology of Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.Peer reviewe

RUVBL1 and XPO1 restrict viral infection in mammalian cells.

<p><b>A–D.</b> Human U2OS cells were transfected with siRNAs against a control, hRuvBL1, or hXPO1 and challenged 3 days post transfection with WNV-KUN for 20 hours (<b>A–B</b>) or VSV for 12 hours (<b>C–D</b>). Cells were fixed, processed for microscopy and quantified in <b>A, C</b>. Mean ± SD of fold change compared to control for 3 independent experiments; * p<0.05, **p<0.01. Cells were processed for northern blots and quantified displaying the mean for 3 independent experiments with control set to 1; * p<0.05, **p<0.01 in <b>B, D</b>. <b>E</b>. 293T cells were transfected with siRNAs against control or two independent siRNAs against hTIP60 and challenged 3 days post transfection with WNV for 24 hours and processed by flow cytometry. Three independent experiments were quantified; Mean ± SD of the fold change in infection is shown and normalized to the control; **p<0.01. <b>F</b>. Primary neurons transduced with lentiviruses expressing the indicated shRNAs were infected with WNV for 24 hours and processed for viral yield by focus forming assays. Mean ± SD for 3 independent experiments; * p<0.05, **p<0.01. <b>G–H</b>. U2OS cells were treated with vehicle or LMB and infected with (<b>G</b>) WNV-KUN or (<b>H</b>) VSV. Mean ± SD of fold change in percent infection compared to control (vehicle) for 3 independent experiments; * p<0.05, ** p<0.01.</p

dXPO1 has antiviral activity in insects.

<p><b>A.</b> Representative images of <i>Drosophila</i> cells treated with control (β-gal) or dXPO1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). <b>B</b>. Quantification of fold change in infection for dsRNA treated cells as in <b>A</b>. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. <b>C–D</b>. Viral RNA levels measured using RT-qPCR in Drosophila cells treated with β-gal (control) or dXPO1 dsRNA and infected with WNV (<b>C</b>) or VSV (<b>D</b>). Mean ± SD of fold change for 3 independent experiments; * p<0.05. <b>E–H</b>. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (<b>E, G</b>) or VSV (<b>F, H</b>) and mortality (<b>E, F</b>) was monitored as a function of time post-infection (** p<0.01 log rank). (<b>G, H</b>) Groups of 15 flies of the indicated genotypes were challenged, and viral titer was assessed by plaque assay in 3 or 4 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean. <b>I–J</b>. Aag2 cells were treated with the indicated dsRNA and then infected with (<b>I</b>) WNV-KUN or (<b>J</b>) VSV. Mean ± SD of fold change in percent infection compared to control (β-gal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.</p

Tip60 complex has antiviral activity.

<p><b>A.</b> Table of RUVBL1-associated complexes, whether the complex is dependent on RUVBL2, and other genes in the complexes tested for antiviral activity. Genes in red were found to be antiviral against both WNV and VSV. <b>B–C</b>. DL1 cells were treated with the indicated dsRNA and then infected with (<b>B</b>) WNV or (<b>C</b>) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01. <b>D–E</b>. Aag2 cells were treated with the indicated dsRNA and then infected with (<b>D</b>) WNV-KUN or (<b>E</b>) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.</p

dRUVBL1 is a broadly antiviral gene.

<p><b>A.</b> Representative images of <i>Drosophila</i> cells treated with control (β-gal) or dRUVBL1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). <b>B</b>, Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. <b>C–D</b>. Viral RNA levels measured using qRT-PCR in <i>Drosophila</i> cells treated with β-gal (control) or dRUVBL1 dsRNA infected with WNV (<b>C</b>) or VSV (<b>D</b>) Mean ± SD of fold change for 3 independent experiments; ** p<0.01. <b>E–H</b>. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (<b>E–F</b>) or VSV (<b>G–H</b>). Mortality was monitored as a function of time post-infection (<b>E,G)</b> (log rank: * p<0.05, ** p<0.01). (<b>F,H)</b> Groups of 15 flies of the indicated genotypes were challenged, and viral titers were assessed by plaque assay in 4–7 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean.</p

Genome-wide RNAi screen in <i>Drosophila</i> for host factors involved in WNV infection.

<p><b>A.</b> Representative images of DL1 cells treated with the indicated dsRNAs and infected with WNV (nuclei, blue; WNV NS1, green). <b>B</b>. Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; ** p<0.01. <b>C</b>. Schematic of screening pipeline including the scatter plot of Robust Z-scores for each gene assayed in duplicate. VSFs (376) and VRFs (161) are noted. <b>D</b>. Bioinformatics show fraction of candidate genes that have human or mosquito orthologs. Significant enrichment of conserved genes (p<0.0001) and under-enrichment of <i>Drosophila</i>-specific genes (p<0.0001) as analyzed by chi-squared test. <b>E</b>. Pie chart of candidate genes and validation results (50 VRF, 96 VSF, 71 not validated). <b>F–G</b>. Gene ontology enrichment of validated genes with five or more members displayed (p<0.001). <b>F</b>. VSF categories enriched. <b>G</b>. VRF categories enriched.</p