title: A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.
abstract: Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, 1142, New Zealand.|School of Biological Sciences, University of Auckland, P.O. Box 92019, Auckland, New Zealand.
authors.forename: Arnaud G
authors.initials: AG
authors.lastname: Blouin
authors.affiliation: School of Biological Sciences, University of Auckland, P.O. Box 92019, Auckland, New Zealand.
authors.forename: Howard A
authors.initials: HA
authors.lastname: Ross
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Qld, 4072, Australia.
authors.forename: Jody
authors.initials: J
authors.lastname: Hobson-Peters
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Qld, 4072, Australia.
authors.forename: Caitlin A
authors.initials: CA
authors.lastname: O'Brien
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, 1142, New Zealand.
authors.forename: Ben
authors.initials: B
authors.lastname: Warren
authors.affiliation: The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, 1142, New Zealand.|School of Biological Sciences, University of Auckland, P.O. Box 92019, Auckland, New Zealand.
authors.forename: Robin
authors.initials: R
authors.lastname: MacDiarmid
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000070357:Asparagaceae / Q000821:virology; D002498:Centrifugation; D007118:Immunoassay / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D009520:New Zealand; D010942:Plant Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D031804:Rumex / Q000821:virology; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000821:virology; D014773:Virology / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D012330:RNA, Double-Stranded; D012367:RNA, Viral
keywords: NGS; antibodies; dsRNA; metagenomics; plant virus