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.

Virological and Immunological Outcomes of Coinfections

“…Microarray-based methods, such as ViroChip (96)(97)(98)(99)(100) and PathChip (101,102), that allowed detection of multiple agents but did not support detection of a divergent virus were developed. Sequenceindependent amplification techniques (next-generation sequencing [NGS] platforms) have been successfully employed for rapid detection of novel (16)(17)(18)(19) and multiple (103) viruses in clinical settings (104)(105)(106) and have allowed whole-virus genome organization (107) and analyses of minority variants (108,109). NGS detects sequences from almost all potential organisms in an unbiased manner.…”

“…In a given clinical specimen, NGS reveals both viral and cellular sequences, but patient privacy must be maintained before transmitting the data from research into clinic settings (103). Nevertheless, the cost of NGS is sharply declining, and in the future it may be competitive with current diagnostic assays (107).…”

Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing

“…There are different dsRNA extraction methods available for HTS. We used the dsRNA extraction kit as it consumes less time (<1 hour), uses less milligrams (50 to 200) of plant tissue as starting material, nor does it require a PCR amplification step that would be required for other dsRNA extraction methods [53][54][55]. In addition, it is very cost-effective per sample.…”

De novo Genome Assembly and Single Nucleotide Variations for Soybean Mosaic Virus Using Soybean Seed Transcriptome Data

“…Therefore, virus-specific sequencing usually requires a purification step for NGS. For example, extraction of double-stranded RNAs from virus-infected organisms followed by NGS is one of the efficient approaches to identify viruses ( Yanagisawa et al, 2016 ). Moreover, sequencing of small RNAs is an alternative technique for virus identification and genome assembly ( Vodovar et al, 2011 ).…”