Archives of Virology

, Volume 157, Issue 2, pp 271–284 | Cite as

Multiple polyadenylated RNA viruses detected in pooled cultivated and wild plant samples

  • Stephen J. WylieEmail author
  • Hao Luo
  • Hua Li
  • Michael G. K. Jones
Original Article


RNA extracted from 120 leaf specimens from 17 plant species was pooled, and polyadenylated RNA species were sequenced together without barcoding in one lane using massively parallel sequencing technology. After analysis, complete or partial genome sequences representing 20 virus isolates of 16 polyadenylated RNA species were identified. In three cases, 2-3 distinct isolates of a virus species co-infected the same plant. Twelve of the viruses identified were described previously and belonged to the genera Potyvirus, Nepovirus, Allexivirus, and Carlavirus. Four were unknown and are proposed as members of the genera Potyvirus, Sadwavirus, and Trichovirus. Virus sequences were subsequently matched to original host plants using RT-PCR assays.


Coat Protein Bean Common Mosaic Virus Apple Chlorotic Leaf Spot Virus Onion Yellow Dwarf Virus Leek Yellow Stripe Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was funded by an Australian Research Council Linkage Grant (LP110200180) and the Murdoch University Institutes of Sustainable Ecosystems, and Crop and Plant Research. Thanks to Professor Kingsley Dixon and Mr. Steve Easton, Botanic Gardens and Parks Authority, for authorizing collections at Kings Park and for assistance in collecting and identifying the plants used from there.

Supplementary material

705_2011_1166_MOESM1_ESM.doc (108 kb)
Supplementary material 1 (DOC 108 kb)


  1. 1.
    Adams MJ, Antoniw JF, Bar-Joseph M, Brunt AA, Candresse T, Foster GD, Martelli GP, Milne RG, Fauquet CM (2004) The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Arch Virol 149:1045–1060PubMedGoogle Scholar
  2. 2.
    Adams MJ, Antoniw JF, Fauquet CM (2005) Molecular criteria for genus and species discrimination within the family Potyviridae. Arch Virol 150:459–479PubMedCrossRefGoogle Scholar
  3. 3.
    Adams IP, Glover RH, Monger WA, Mumford R, Jackeviciene E, Navalinskiene M, Samuitiene M, Boonham N (2009) Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology. Mol Plant Pathol 10:537–545PubMedCrossRefGoogle Scholar
  4. 4.
    Al Rwahnih M, Daubert S, Golino D, Rowhani A (2009) Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. Virology 387:395–401PubMedCrossRefGoogle Scholar
  5. 5.
    Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  6. 6.
    Asjes CJ (1998) Data Sheet on Lily Mottle Potyvirus for CABI Crop Protection Compendium. CABI Information. Wallingford, OxonGoogle Scholar
  7. 7.
    Balijja A, Kvarnheden A, Turchetti T (2008) A non-phenol–chloroform extraction of double-stranded RNA from plant and fungal tissues. J Virol Methods 152:32–37PubMedCrossRefGoogle Scholar
  8. 8.
    Blouin AG, Greenwood DR, Chavan RR, Pearson MN, Clover GRG, MacDiarmid RM, Cohen D (2010) A generic method to identify plant viruses by high-resolution tandem mass spectrometry of their coat proteins. J Virol Methods 163:49–56PubMedCrossRefGoogle Scholar
  9. 9.
    Boonham N, Adams I, Glover R, Monger W, Hodges T, Ashton P (2010) High throughput sequencing—next wave diagnostics. Phytopathology 100(suppl. 1):S154Google Scholar
  10. 10.
    Carstens EB (2010) Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2009). Arch Virol 155:133–146PubMedCrossRefGoogle Scholar
  11. 11.
    Chen J, Zheng HY, Antoniw JF, Adams MJ, Chen JP, Lin L (2004) Detection and classification of allexiviruses from garlic in China. Arch Virol 149:435–445PubMedCrossRefGoogle Scholar
  12. 12.
    Chung BYW, Miller WA, Atkins JF, Firth AE (2008) An over-lapping essential gene in the Potyviridae. Proc Natl Acad Sci USA 105:5897–5902PubMedCrossRefGoogle Scholar
  13. 13.
    Coetzee B, Freeborough M-J, Maree HJ, Celton J-M, Rees DJG, Burger JT (2010) Deep sequencing analysis of viruses infecting grapevines: virome of a vineyard. Virology 400:157–163PubMedCrossRefGoogle Scholar
  14. 14.
    Conci VC, Canavelli AE, Balzarini MG (2010) The distribution of garlic viruses in leaves and bulbs during the first year of infection. J Phytopath 158:186–193CrossRefGoogle Scholar
  15. 15.
    Donaire L, Wang Y, Gonzalez-Ibeas D, Mayer KF, Aranda MA, Llave C (2009) Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 392:203–214PubMedCrossRefGoogle Scholar
  16. 16.
    Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011) Geneious v5.4. Accessed 15 June 2011
  17. 17.
    Eisen JA (2007) Environmental shotgun sequencing: its potential and challenges for studying the hidden world of microbes. PLoS Biol 5:e82PubMedCrossRefGoogle Scholar
  18. 18.
    Gibbs AJ, Mackenzie AM, Wei KJ, Gibbs MJ (2008) The potyviruses of Australia. Arch Virol 153:1411–1420PubMedCrossRefGoogle Scholar
  19. 19.
    Gorbalenya AE, Donchenko AP, Blinov VM, Koonin EV (1989) Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. FEBS Lett 243:103–114PubMedCrossRefGoogle Scholar
  20. 20.
    Gorbalenya AE, Koonin EV, Wolf YI (1990) A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses. FEBS Lett 262:145–148PubMedCrossRefGoogle Scholar
  21. 21.
    Hagen C, Frizzi A, Kao J, Jia L, Huang M, Zhang Y, Huang S (2011) Using small RNA sequences to diagnose, sequence, and investigate the infectivity characteristics of vegetable-infecting viruses. Arch Virol 156:1209–1216PubMedCrossRefGoogle Scholar
  22. 22.
    Haible D, Kober S, Jeske H (2006) Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses. J Virol Methods 135:9–16PubMedCrossRefGoogle Scholar
  23. 23.
    Hanada K, Fukumoto F, Kusunoki M, Kameya-Iwaki M, Tanaka Y, Iwanami T (2006) Cycas necrotic stunt virus isolated from gladiolus plants in Japan. J Gen Plant Pathol 72:383–386CrossRefGoogle Scholar
  24. 24.
    Hiramatsu M, Ii K, Okubo H, Huang K-L, Huang C-W (2001) Biogeography and origin of Lilium longiflorum and L. formosanum (Liliaceae) endemic to the Ryukyu Archipelago and Taiwan as determined by allozyme diversity. Am J Bot 88:1230–1239PubMedCrossRefGoogle Scholar
  25. 25.
    Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7PubMedCrossRefGoogle Scholar
  26. 26.
    Kusunoki M, Hanada K, Iwaki M, Chang MU, Doi Y, Yora K (1986) Cycas necrotic stunt virus, a new member of nepoviruses found in Cycas revolute-Host range, purification, serology and some other properties. Ann Phytopathol Soc Japan 52:302–311CrossRefGoogle Scholar
  27. 27.
    Le Gall O, Iwanami T, Jones AT, Lehto K, Sanfacon H, Wellink J, Wetzel T, Yoshikawa N (2005) Comoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) Virus taxonomy, eighth report of the international committee on the taxonomy of viruses. Elsevier Academic Press, London, pp 807–818Google Scholar
  28. 28.
    Le Gall O, Sanfaçon H, Ikegami M, Iwanami T, Jones T, Karasev A, Lehto K, Wellink J, Wetzel T, Yoshikawa N (2007) Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae). Arch Virol 152:1767–1774PubMedCrossRefGoogle Scholar
  29. 29.
    Luo H, Wylie SJ, Jones MGK (2010) Identification of plant viruses using one-dimensional gel electrophoresis and peptide mass fingerprints. J Virol Meth 165:297–301CrossRefGoogle Scholar
  30. 30.
    Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–48PubMedGoogle Scholar
  31. 31.
    Martelli GP, Adams MJ, Kreuze JF, Dolja VV (2007) Family Flexiviridae: a case study in virion and genome plasticity. Ann Rev Phytopathol 45:73–100CrossRefGoogle Scholar
  32. 32.
    Mayo MA, Fritsch C (1994) A possible consensus sequence for VPg of viruses in the family Comoviridae. FEBS Lett 354:129–130PubMedCrossRefGoogle Scholar
  33. 33.
    Melcher U, Muthukumar V, Wiley GB, Min BE, Palmer MW, Verchot-Lubicz J, Ali A, Nelson RS, Roe BA, Thapa V, Pierce ML (2008) Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya. J Virol Meth 152:49–55CrossRefGoogle Scholar
  34. 34.
    Morschel JR (1966) Recorded plant diseases in and outside australia: part 4–forest trees and ornamental plants. Commonwealth Department of Health Division of Plant Quarantine, CanberraGoogle Scholar
  35. 35.
    Mushegian AR (1994) The putative movement domain encoded by nepovirus RNA-2 is conserved in all sequenced nepoviruses. Arch Virol 135:437–441PubMedCrossRefGoogle Scholar
  36. 36.
    Ochoa-Corona FM, Elliot DR, Tang Z, Lebas BSM, Alexander BJR (2003) Detection of Cycas necrotic stunt virus (CNSV) in post-entry quarantine stocks of ornamentals in New Zealand. Phytopathology 93:S67Google Scholar
  37. 37.
    Remah A, Jones AT, Mitchell MJ (1986) Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia. Ann Appl Biol 109:307–315CrossRefGoogle Scholar
  38. 38.
    Riechmann JL, Lain S, Garcia JA (1992) Highlights and prospects of potyvirus molecular biology. J Gen Virol 73:1–16PubMedCrossRefGoogle Scholar
  39. 39.
    Ritzenthaler C, Viry M, Pinck M, Margis R, Margis R, Fuchs M, Pinck L (1991) Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1. J Gen Virol 72:2357–2365PubMedCrossRefGoogle Scholar
  40. 40.
    Roossinck MJ, Saha P, Wiley GB, Quan J, White JD, Lai H, Chavarŕia F, Shen G, Roe BA (2010) Ecogenomics: using massively parallel pyrosequencing to understand virus ecology. Mol Ecol 19(Suppl. 1):81–88PubMedCrossRefGoogle Scholar
  41. 41.
    Rott ME, Gilchrist A, Lee L, Rochon DM (1995) Nucleotide sequence of tomato ringspot virus RNA1. J Gen Virol 76:465–471PubMedCrossRefGoogle Scholar
  42. 42.
    Sanfaçon H, Wellink J, Le Gall O, Karasev A, van der Vlugt R, Wetzel T (2009) Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus. Arch Virol 154:899–907PubMedCrossRefGoogle Scholar
  43. 43.
    Shukla DD, Ward CW, Brunt AA (1994) The Potyviridae. CAB International, WallingfordGoogle Scholar
  44. 44.
    Sward RJ (1990) Lettuce necrotic yellows rhabdovirus and other viruses infecting garlic. Australas Plant Pathol 19:46–51CrossRefGoogle Scholar
  45. 45.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. doi: 10.1093/molbev/msr121
  46. 46.
    Tsuneyoshi T, Matsumi T, Natsuaka KT, Sumi S (1998) Nucleotide sequence analysis of virus isolates indicates the presence of three potyvirus species in Allium plants. Arch Virol 143:97–113PubMedCrossRefGoogle Scholar
  47. 47.
    Webster C, Jones RAC, Coutts BA, Jones MGK, Wylie SJ (2007) Virus impact at the interface of an ancient ecosystem and a recent agroecosystem: studies on three legume-infecting potyviruses in the southwest Australian floristic region. Plant Pathol 56:729–742CrossRefGoogle Scholar
  48. 48.
    Westphal MI, Browne M, MacKinnon K, Noble I (2008) The link between international trade and the global distribution of invasive alien species. Biol Invasions 10:391–398CrossRefGoogle Scholar
  49. 49.
    Wren JD, Roossinck MJ, Nelson RS, Scheets K, Palmer MW, Melcher U (2006) Plant virus biodiversity and ecology. PLoS Biol 4:e80PubMedCrossRefGoogle Scholar
  50. 50.
    Wu Q, Luo Y, Lu R, Lau N, Lai EC, Li WX, Ding SW (2010) Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs. Proc Natl Acad Sci USA 107:1606–1611PubMedCrossRefGoogle Scholar
  51. 51.
    Wylie SJ, Jones MGK (2011) Characterisation and quantitation of mutant and wild-type genomes of Hardenbergia mosaic virus isolates co-infecting a wild plant of Hardenbergia comptoniana. Arch Virol 156:1287–1290PubMedCrossRefGoogle Scholar
  52. 52.
    Wylie SJ, Jones MGK (2011) Hardenbergia virus A, a novel member of the Betaflexiviridae from a wild legume in South-west Australia. Arch Virol 156:1245–1250PubMedCrossRefGoogle Scholar
  53. 53.
    Wylie SJ, Jones MGK (2011) The complete genome sequence of Passionfruit woodiness virus determined using deep sequencing, and its relationship to other potyviruses. Arch Virol 156:479–482PubMedCrossRefGoogle Scholar
  54. 54.
    Yan F, Zhang HM, Adams MJ, Yang J, Peng JJ, Antoniw JF, Zhou YJ, Chen JP (2010) Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing. Arch Virol 155:935–940PubMedCrossRefGoogle Scholar
  55. 55.
    Yeh SD, Gonsalves D, Wang HL, Namba R (1988) Control of papaya ringspot virus by cross protection. Plant Dis 72:375–380CrossRefGoogle Scholar
  56. 56.
    Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829PubMedCrossRefGoogle Scholar
  57. 57.
    Zheng HY, Chen J, Zhao MF, Lin L, Chen JP, Antoniw JF, Adams MJ (2003) Occurrence and sequences of Lily mottle virus and Lily symptomless virus in plants grown from imported bulbs in Zhejiang province, China. Arch Virol 148:2419–2428PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Stephen J. Wylie
    • 1
    Email author
  • Hao Luo
    • 1
  • Hua Li
    • 1
  • Michael G. K. Jones
    • 1
  1. 1.Plant Virology Group, Western Australian State Agricultural Biotechnology Centre, School of Biological Sciences and BiotechnologyMurdoch UniversityPerthAustralia

Personalised recommendations