title: Discovery of DNA viruses in wild-caught mosquitoes using small RNA high throughput sequencing.
abstract: BACKGROUND
  Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens.
  
  
  RESULTS
  In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18-30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/- strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5'- and 3' -untranslated regions where no transcripts were expected.
  
  
  CONCLUSIONS
  Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors.
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors.forename: Maijuan
authors.initials: M
authors.lastname: Ma
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Zhengda
authors.initials: Z
authors.lastname: Gong
authors.forename: Lu
authors.initials: L
authors.lastname: Zhuang
authors.forename: Cun
authors.initials: C
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.forename: Yigang
authors.initials: Y
authors.lastname: Tong
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Wuchun
authors.initials: W
authors.lastname: Cao
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D009033:Culicidae / Q000821:virology*; D017990:Densovirus / Q000235:genetics* / Q000302:isolation & purification; D016679:Genome, Viral / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D022661:RNA, Untranslated / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012015:Reference Standards; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D022661:RNA, Untranslated; D012367:RNA, Viral