A metagenomic approach using next-generation sequencing for viral profiling of a vineyard and genetic characterization of grapevine virus E

[摘要] ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largelydue to the high sequence data throughput capacity and unbiased approach in determining thegenetic composition of an unknown environmental sample. This study investigated theapplicability of the Illumina next-generation sequencing platform for metagenomic sequencingof grapevine viruses to provide the first complete viral profile, or virome, of a diseasedvineyard.Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard inSouth Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA wassequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesistechnique, and more than 19 million sequence reads, equivalent to approximately 837megabases of metagenomic sequence data, were obtained. Of these data, approximately 400megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. Thesescaffolds were subjected to BLAST searches against the NCBI databases and top hit scores wereused for virus identification. Based on the BLAST results, suitable sequences were selected fromthe NCBI database and used as reference sequence in MAQ mapping assemblies.The bioinformatic analyses allowed for the determination of the virus species present, the mostprominent variants, and the relative abundance of each. Four known grapevine viral pathogenswere identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed shortread sequence data, was identified as the most prominent virus species. Three variants of thisvirus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant anda potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stempitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member ofmolecular group III, were identified. This study is also the first to report the presence ofGrapevine virus E (GVE) in South African vineyards.Grapevine virus E was further genetically characterized and the genome sequence of GVEisolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is thefirst complete genome sequence for the virus species. The genome organization of GVE SA94 istypical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within thehelicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100%nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generatedfrom the metagenomic sequence data.Bioinformatic analysis of metagenomic sequence data further revealed the presence of threefungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus,Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar toPenicillium chrysogenum virus, was the second most abundant virus detected.We demonstrated the successful application of a short read sequencing technology, such as theIllumina platform, for viral profiling of an infected vineyard. To our knowledge this is the firstapplication of the Illumina technology for this purpose.

[发布机构] Stellenbosch University