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      Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

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          Abstract

          In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.

          Author Summary

          Mosquitoes of the Aedes family transmit many important viruses, including dengue virus, between their vertebrate hosts. In the mosquito, the growth of these viruses is limited by the antiviral RNA interference pathway. Key to this pathway is a class of small non-coding RNAs known as small interfering RNAs (siRNAs). In addition, two related but distinct small RNA pathways known as the microRNA (miRNA) and the PIWI-interacting RNA (piRNA) pathway are implicated in regulating virus replication in mosquitoes. Thus, since small RNAs may critically influence the transmission of dengue virus, we set out to analyze the populations of viral and mosquito small RNAs that are produced in infected Aedes mosquito cells. We found that besides the well-known viral siRNAs, dengue virus-derived piRNAs were produced in these cells and we identified the PIWI proteins that these small RNAs rely on. In addition, we found that viral miRNAs were not expressed from the dengue virus genome and that the levels of mosquito miRNAs were barely changed upon infection. Finally, our data allowed for the identification of novel Aedes miRNAs, complementing the repertoire of these important regulatory RNAs in vector mosquitoes.

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          Most cited references 39

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          A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila.

          In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI.
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            Manipulation of FASTQ data with Galaxy

            Summary: Here, we describe a tool suite that functions on all of the commonly known FASTQ format variants and provides a pipeline for manipulating next generation sequencing data taken from a sequencing machine all the way through the quality filtering steps. Availability and Implementation: This open-source toolset was implemented in Python and has been integrated into the online data analysis platform Galaxy (public web access: http://usegalaxy.org; download: http://getgalaxy.org). Two short movies that highlight the functionality of tools described in this manuscript as well as results from testing components of this tool suite against a set of previously published files are available at http://usegalaxy.org/u/dan/p/fastq Contact: james.taylor@emory.edu; anton@bx.psu.edu Supplementary information: Supplementary data are available at Bioinformatics online.
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              RNA-based antiviral immunity.

               Wei-Qun Ding (2010)
              In eukaryotic RNA-based antiviral immunity, viral double-stranded RNA is recognized as a pathogen-associated molecular pattern and processed into small interfering RNAs (siRNAs) by the host ribonuclease Dicer. After amplification by host RNA-dependent RNA polymerases in some cases, these virus-derived siRNAs guide specific antiviral immunity through RNA interference and related RNA silencing effector mechanisms. Here, I review recent studies on the features of viral siRNAs and other virus-derived small RNAs from virus-infected fungi, plants, insects, nematodes and vertebrates and discuss the innate and adaptive properties of RNA-based antiviral immunity.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                25 February 2016
                February 2016
                : 10
                : 2
                Affiliations
                [1 ]Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands
                [2 ]Centre for Immunity, Infection & Evolution, University of Edinburgh, Edinburgh, United Kingdom
                Colorado State University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: PM RPvR. Performed the experiments: PM. Analyzed the data: PM AI AHB RPvR. Wrote the paper: PM AI AHB RPvR. Performed bioinformatic analyses: PM AI.

                Article
                PNTD-D-15-01748
                10.1371/journal.pntd.0004452
                4767436
                26914027
                © 2016 Miesen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 6, Tables: 1, Pages: 22
                Product
                Funding
                This work is financially supported by a PhD fellowship from Radboud University Medical Center ( www.radboudumc.nl) to PM, a Strategic award from the Wellcome Trust to the Centre for Infection Immunity and Evolution (grant no. 095831), Welcome Trust (grant no. WT097394A1A) to AHB, an ECHO project grant from the Netherlands Organization for Scientific Research (NWO, grant no. 711.013.001) to RPvR, and European Research Council Consolidator Grant under the European Union’s Seventh Framework Programme (ERC, grant no. 615680) to RPvR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                MicroRNAs
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                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
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                Biochemistry
                Nucleic acids
                RNA
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                Medicine and Health Sciences
                Epidemiology
                Disease Vectors
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                Custom metadata
                Small RNA sequences have been submitted to NCBI Sequence Read Archive under accession numbers SRX1309506 to SRX1309511. All other relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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