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

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 detecti...

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Autores principales: Yanagisawa, Hironobu, Tomita, Reiko, Katsu, Koji, Uehara, Takuya, Atsumi, Go, Tateda, Chika, Kobayashi, Kappei, Sekine, Ken-Taro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810260/
https://www.ncbi.nlm.nih.gov/pubmed/27072419
http://dx.doi.org/10.3390/v8030070
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author Yanagisawa, Hironobu
Tomita, Reiko
Katsu, Koji
Uehara, Takuya
Atsumi, Go
Tateda, Chika
Kobayashi, Kappei
Sekine, Ken-Taro
author_facet Yanagisawa, Hironobu
Tomita, Reiko
Katsu, Koji
Uehara, Takuya
Atsumi, Go
Tateda, Chika
Kobayashi, Kappei
Sekine, Ken-Taro
author_sort Yanagisawa, Hironobu
collection PubMed
description 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.
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spelling pubmed-48102602016-04-04 Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses Yanagisawa, Hironobu Tomita, Reiko Katsu, Koji Uehara, Takuya Atsumi, Go Tateda, Chika Kobayashi, Kappei Sekine, Ken-Taro Viruses Communication 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. MDPI 2016-03-07 /pmc/articles/PMC4810260/ /pubmed/27072419 http://dx.doi.org/10.3390/v8030070 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Yanagisawa, Hironobu
Tomita, Reiko
Katsu, Koji
Uehara, Takuya
Atsumi, Go
Tateda, Chika
Kobayashi, Kappei
Sekine, Ken-Taro
Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title_full Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title_fullStr Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title_full_unstemmed Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title_short Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses
title_sort combined decs analysis and next-generation sequencing enable efficient detection of novel plant rna viruses
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810260/
https://www.ncbi.nlm.nih.gov/pubmed/27072419
http://dx.doi.org/10.3390/v8030070
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