The double-stranded (ds) RNA enrichment tool based on antibody recognition of this uncommon RNA structure was very effective for the detection of highly replicating viruses, mostly from herbaceous plants as demonstrated in the publication of Blouin et al. (2016). Following an initial optimisation round to recover sufficient virus nucleic acid in the smallest possible volume of sample processing buffer, a method was established to trial virus enrichment on a set of grapevine samples. The grapevine samples were accordingly processed then subjected to NGS (Illumina HiSeq, 125 bp paired end reads) to determine the success of the sample preparation method and the enrichment for dsRNA. However, the majority of the sequences obtained were of plant origin, in particular matching the ribosomes. Therefore, when applied to grapevines, the tool was not sufficiently sensitive; it enriched for ribosomal RNA (rRNA) rather than virus-derived dsRNA. We therefore further improved the virus dsRNA enrichment method from such recalcitrant, woody perennial plant tissue with low titre viruses restricted to phloem tissue. First we developed a Taqman assay to determine the concentration of virus-derived and rRNA captured by the antibodies, then trialled a range of variables and additives in the enrichment protocol to achieve the highest amount of virus-derived dsRNA and the lowest amount of rRNA dsRNA prior to NGS. The improved method for virus enrichment from intractable tissues resulted in an increased viral enrichment in excess of 10,000 fold of the original method. When sequenced, the preparation enabled the discovery of a new-to-science virus that adds to the well described grapevine virome. The tool will be of great use for metagenomics of woody perennial plants or other species that typically support only low virus titres and thus pose a challenge for virus enrichment and identification.
Blouin et al., 2016. Molecular Ecology Resources 16:1255-63.