title: Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.
abstract: The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses.
authors.affiliation: NARO Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. yana1208@affrc.go.jp.|Yokohama Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries, 1-7 Nagamine, Tsukuba, Ibaraki 305-0052, Japan. yana1208@affrc.go.jp.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Yanagisawa
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. r-tomita@ibrc.or.jp.
authors.forename: Reiko
authors.initials: R
authors.lastname: Tomita
authors.affiliation: Yokohama Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries, 1-7 Nagamine, Tsukuba, Ibaraki 305-0052, Japan. katsuk@pps.maff.go.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Katsu
authors.affiliation: Yokohama Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries, 1-7 Nagamine, Tsukuba, Ibaraki 305-0052, Japan. ueharat@pps.maff.go.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Uehara
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. go-atsumi@aist.go.jp.|National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamuhigashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan. go-atsumi@aist.go.jp.
authors.forename: Go
authors.initials: G
authors.lastname: Atsumi
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. c-tateda@ibrc.or.jp.
authors.forename: Chika
authors.initials: C
authors.lastname: Tateda
authors.affiliation: Laboratory of Plant Molecular Biology and Virology, Faculty of Agriculture, Ehime University, 3-5-7, Tarumi, Matsuyama, Ehime 790-8566, Japan. kappei@ehime-u.ac.jp.
authors.forename: Kappei
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan. k-sekine@ibrc.or.jp.|Laboratory of Plant Pathology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan. k-sekine@ibrc.or.jp.
authors.forename: Ken-Taro
authors.initials: KT
authors.lastname: Sekine
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D010944:Plants / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*
chemical_list: D012330:RNA, Double-Stranded
keywords: Blueberry shoestring virus; Sobemovirus; blueberry; deep-sequencing; detection; dsRNA; dsRNA-binding protein