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Detection of Plant Viruses in Natural Environments by Using RNA-Seq

Part of the Methods in Molecular Biology book series (MIMB,volume 1236)


Sequencing of RNA by next generation sequencers, RNA-Seq, is revolutionizing virus detection. In addition to the unbiased detection of various viruses from wild plants in natural environments, RNA-Seq also allows for the parallel collection of host plant transcriptome data. Host transcriptome data are highly valuable for studying the responses of hosts to viral infections, as well as viral host manipulation. When detecting viruses using RNA-Seq, it is critical to choose appropriate methods for the removal of rRNA from total RNA. Although viruses with polyadenylated genomes can be detected by RNA-Seq following mRNA purification using oligo-dT beads, viruses with non-polyadenylated genomes are not effectively detected. However, such viruses can be detected by RNA-Seq using the rRNA selective depression method. The high-throughput and cost-effective method of RNA-Seq library preparation which is described here allows us to detect a broad range of viruses in wild plants.

Key words

  • Virus detection
  • RNA-Seq
  • Wild plant
  • Selective depression of rRNA
  • Natural environment

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  1. Hull R (2002) Matthews’ plant virology, 4th edn. Elsevier, Amsterdam

    Google Scholar 

  2. Malmstrom CM, Melcher U, Bosque-Pérez NA (2011) The expanding field of plant virus ecology: historical foundations, knowledge gaps, and research directions. Virus Res 159:84–94

    PubMed  CrossRef  CAS  Google Scholar 

  3. Le Guyader F, Dubois E, Menard D, Pommepuy M (1994) Detection of Hepatitis-A virus, rotavirus, and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested PCR. Appl Environ Microbiol 60(10):3665–3671

    PubMed  PubMed Central  Google Scholar 

  4. Munster VJ, Baas C, Lexmond P, Waldenstrom J, Wallensten A, Fransson T et al (2007) Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds. PLoS Pathog 3:e61

    PubMed  CrossRef  PubMed Central  Google Scholar 

  5. Dovas CI, Papanastassopoulou M, Georgiadis MP, Chatzinasiou E, Maliogka VI, Georgiades GK (2010) Detection and quantification of infectious avian influenza A (H5N1) Virus in environmental water by using real-time reverse transcription-PCR. Appl Environ Microbiol 76:2165–2174

    PubMed  CrossRef  CAS  PubMed Central  Google Scholar 

  6. Honjo MN, Minamoto T, Kawabata Z (2012) Reservoirs of Cyprinid herpesvirus 3 (CyHV-3) DNA in sediments of natural lakes and ponds. Vet Microbiol 155:183–190

    PubMed  CrossRef  CAS  Google Scholar 

  7. Fargette D, Fauquet C, Grenier E, Thresh J (1990) The spread of African cassava mosaic-virus into and within cassava fields. J Phytopathol 130:289–302

    CrossRef  Google Scholar 

  8. Roossinck MJ (2012) Plant virus metagenomics: biodiversity and ecology. Annu Rev Genet 46:359–369

    PubMed  CrossRef  CAS  Google Scholar 

  9. Nagano AJ, Sato Y, Mihara M, Antonio BA, Motoyama R, Itoh H et al (2012) Deciphering and prediction of transcriptome dynamics under fluctuating field conditions. Cell 151:1358–1369

    PubMed  CrossRef  CAS  Google Scholar 

  10. Wang L, Si Y, Dedow LK, Shao Y, Liu P, Brutnell TP (2011) A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq. PLoS One 6(10):e26426

    PubMed  CrossRef  CAS  PubMed Central  Google Scholar 

  11. Zhong S, Joung JG, Zheng Y, Chen YR, Liu B, Shao Y et al (2011) High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harb Protoc 2011:940–949

    PubMed  CrossRef  Google Scholar 

  12. Morlan JD, Qu K, Sinicropi DV (2012) Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One 7:e42882

    PubMed  CrossRef  CAS  PubMed Central  Google Scholar 

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This work was supported by JST PRESTO (to A.J.N.) and the Next Program (GS013) (to H.K.).

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Correspondence to Atsushi J. Nagano or Hiroshi Kudoh .

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Nagano, A.J., Honjo, M.N., Mihara, M., Sato, M., Kudoh, H. (2015). Detection of Plant Viruses in Natural Environments by Using RNA-Seq. In: Uyeda, I., Masuta, C. (eds) Plant Virology Protocols. Methods in Molecular Biology, vol 1236. Humana Press, New York, NY.

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  • Print ISBN: 978-1-4939-1742-6

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