Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts Shelley Cook1*☯, Betty Y.-W. Chung2☯, David Bass1, Gregory Moureau3, Shuoya Tang2, Erica McAlister1, C. Lorna Culverwell1, Edvard Glücksman4, Hui Wang5, T. David K. Brown6, Ernest A. Gould3,5, Ralph E. Harbach1, Xavier de Lamballerie3, Andrew E. Firth6* 1 Department of Life Sciences, Natural History Museum, London, United Kingdom, 2 Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom, 3 UMR_D 190 "Emergence des Pathologies Virales" (Aix-Marseille Univ. IRD French Institute of Research for Development EHESP French School of Public Health), Marseille, France, 4 Department of General Botany, University Duisburg-Essen, Essen, Germany, 5 Centre for Ecology & Hydrology, Wallingford, Oxfordshire, United Kingdom, 6 Department of Pathology, University of Cambridge, Cambridge, United Kingdom Abstract We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected. Citation: Cook S, Chung BY-W, Bass D, Moureau G, Tang S, et al. (2013) Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts. PLoS ONE 8(11): e80720. doi:10.1371/journal.pone.0080720 Editor: Houssam Attoui, The Pirbright Institute, United Kingdom Received June 14, 2013; Accepted October 7, 2013; Published November 18, 2013 Copyright: © 2013 Cook 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. Funding: SC was funded by a Wellcome Trust Sir Henry Wellcome Postdoctoral Fellowship [082744]. See www.wellcome.ac.uk. Work in the AEF lab is funded by grants from the Wellcome Trust [088789] and the U.K. Biotechnology and Biological Research Council (BBSRC) [BB/J007072/1 and BB/ J015652/1]. See www.wellcome.ac.uk and www.bbscr.ac.uk respectively. BYWC is funded by an EMBO Long-Term Postdoctoral Fellowship and a Sir Henry Wellcome Postdoctoral Fellowship [096082]. See www.embo.org and www.wellcome.ac.uk respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (SC); [email protected] (AEF) ☯ These authors contributed equally to this work. Introduction research has demonstrated that there exists a vast diversity of previously undiscovered viruses in the natural environment [1]. The emergence of new infectious diseases, many of which For example, a large number of “insect-specific” flaviviruses are caused by RNA viruses, is a major threat to human, animal have been discovered recently in numerous culicine mosquito and plant health and agriculture. RNA viruses demonstrate species (Diptera: Culicidae: Culicinae) and these viral strains remarkable capacity to evolve due to large population size, are likely to vastly outnumber the pathogenic strains present in short generation times and high mutation and recombination the natural environment, including those flaviviruses that cause rates. Many are also vector-borne, potentially increasing the diseases such as yellow fever and dengue fever in humans prevalence and range of a given virus in natural ecosystems. [2,3,4,5]. A variety of other novel RNA and DNA viruses have Understanding the mechanisms underlying viral emergence is been recently identified in insects [6,7]. Deep sequencing key for the rational design of antiviral therapies and control technologies have the potential to provide an unprecedented strategies. A first step in this process is the characterisation of description of this genetic background, and thus to begin to the true distribution of virus genetic variability, both spatially understand the interactions of pathogenic and “silent” viruses in and taxonomically across different host species. Recent nature, which may include competitive exclusion, PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80720 RNA Reconstruction of Novel Viruses from Diptera superinfection, recombination and other mechanisms that may pooling aims was to collect (i) adult anophelines, (ii) adult be involved in the generation of viral diversity [8,9,30]. culicines, (iii) immature anophelines and (iv) immature Invertebrates, including dipterans (order Diptera, the two- culicines, from each collection site, plus (v) a pooled sample of winged flies) such as mosquitoes, respond to viral infection via chironomids from across all sites, for comparison of viral RNA interference (RNAi) or RNA silencing, leading to diversity. suppression or elimination of the pathogen via sequence- specific degradation of homologous RNA sequences into small Total and small RNA preparation, library construction RNAs (sRNAs) of discrete sizes. This has been demonstrated and Illumina sequencing in Stegomyia aegypti [=Aedes aegypti] mosquitoes infected RNA was purified from 0.5 g of flies using the Ambion with dengue virus (DENV) or Sindbis virus (SINV), Culex mirVana miRNA Isolation Kit (PE Applied BioSystems, quinquefasciatus mosquitoes orally exposed to West Nile virus Warrington, England) according to the manufacturer’s (WNV) and Drosophila infected with flock house virus (FHV) instructions for total and small-enriched RNA. Additionally, the [10,11,12,13]. Similarly, studies in RNAi-deficient Anopheles latter were DNase-treated. All RNA extractions were stored at gambiae mosquitoes showed increased viral dissemination -80°C and evaluated on a Bioanalyzer (Agilent Technology, rates and titres of inoculated O’nyong-nyong virus [14]. In this Santa Clara, CA, USA). study, we used sRNAs sequenced from mosquitoes and Twelve tagged small-enriched RNA libraries were prepared chironomids sampled from the natural environment to inform for single read sequencing using the HiSeq 2000 platform total RNA deep sequencing of samples of particular interest. (Illumina, San Diego, CA, USA). Briefly, acrylamide gel Within the mosquitoes, the vast majority of flaviviruses purification of RNA bands corresponding to size range ~20–30 discovered to date have been isolated from culicine mosquito nt was conducted. Adaptor sequences were added so that the species (subfamily Culicinae, see examples above) as libraries could be multiplexed, producing 10–19 million reads opposed to anopheline mosquitoes (subfamily Anophelinae, for per tagged library. Based on the sRNA sequencing, we chose example An. gambiae, a major vector for malarial parasites, three samples for further investigation: sample 1 (140 adult see example above), despite the fact that species from both anopheline mosquitoes, female Anopheles maculipennis sensu groups of Culicidae take bloodmeals. In contrast, the lato, from the farm/stables site), sample 7 (adult chironomids chironomids (Diptera: Chironomidae) are non-biting midges. pooled across sites) and sample 9 (25 adult culicine We planned to sample all three groups in order to test whether mosquitoes, female Ochlerotatus caspius and Oc. detritus from related viruses may be discovered in a range of dipterans in the rice farm). Since genome assembly from sRNA alone is the natural environment, potentially related to a shared difficult due to non-uniform and incomplete coverage and short environment and/or transmission and maintenance cycles sequence reads, we subjected total RNA samples to HiSeq aside from blood-feeding. sequencing in an attempt to assemble full-genome virus sequences. Materials and Methods Sequence processing, assembly and virus genome Trapping protocol identification Approximately 600 mosquitoes and chironomids were Sequence read processing, assembly and virus genome collected in June 2011 in the Camargue region of France for identification were conducted using a custom bioinformatics screening. Specimens were sampled from a variety of locations pipeline. Trimmed sRNA reads were assembled using Velvet using different collection methods to maximise species [16] and contigs were compared to a database comprising all diversity. Collections were made within and around (i) an National Center for Biotechnology Information (NCBI) RNA ornithological park, (ii) a rice farm and (iii) a rural farmhouse virus proteins using blastx [17]. For the total RNA samples, raw and stables. CDC fan-augmented light traps were Illumina reads were trimmed using the FASTX-Toolkit (see supplemented