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Open AccessCommunication
Viruses 2016, 8(3), 70; doi:10.3390/v8030070

Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

1
NARO Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan
2
Yokohama Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries, 1-7 Nagamine, Tsukuba, Ibaraki 305-0052, Japan
3
Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan
4
National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamuhigashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan
5
Laboratory of Plant Molecular Biology and Virology, Faculty of Agriculture, Ehime University, 3-5-7, Tarumi, Matsuyama, Ehime 790-8566, Japan
6
Laboratory of Plant Pathology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan
*
Author to whom correspondence should be addressed.
Academic Editor: Johnson Mak
Received: 30 November 2015 / Revised: 26 January 2016 / Accepted: 8 February 2016 / Published: 7 March 2016
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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. View Full-Text
Keywords: deep-sequencing; dsRNA; dsRNA-binding protein; detection; blueberry; Sobemovirus; Blueberry shoestring virus deep-sequencing; dsRNA; dsRNA-binding protein; detection; blueberry; Sobemovirus; Blueberry shoestring virus
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MDPI and ACS Style

Yanagisawa, H.; Tomita, R.; Katsu, K.; Uehara, T.; Atsumi, G.; Tateda, C.; Kobayashi, K.; Sekine, K.-T. Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses. Viruses 2016, 8, 70.

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