Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. Out of 38 samples analyzed, 37 had mixed infections with 4–11 viruses, indicating a high viral load. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017–2018. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked. Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. The present work highlights the fact that even though HTS technologies produce invaluable data for the description of the sanitary status of a plant, in-depth biological studies are necessary before assigning a new virus to a particular host in such metagenomic approaches. Indeed, the Tymoviridae family comprises isometric viruses infecting a wide range of hosts in different kingdoms (Plantae, Fungi, and Animalia). Description of a New Tymovirales samples at some periods of the year. This virus was never detected in the spring period, raising the possibility that it might not be a grapevine-infecting virus, but rather a virus infecting a grapevine-associated organism that may be transiently present on grapevine Hily et al. Detection of this new agent in the original grapevines proved very erratic and was only consistent at the end of the growing season. Nonetheless, the new virus shows enough differences to be considered as a new species defining a new genus. This genome displays an organization similar to Tymoviridae family members in the Tymovirales order. In addition, a novel viral genome was detected in all four datasets, consisting of a single-stranded, positive-sense RNA molecule of 6033 nucleotides. From four RNAseq datasets, a few complete grapevine-infecting virus and viroid genomes were de-novo assembled: (a) three GFLV genomes, 11 grapevine rupestris stem-pitting associated virus (GRSPaV) and six viroids. In the present study, a total RNAseq-based approach was used to determine the full genome sequences of various grapevine fanleaf virus (GFLV) isolates and to analyze the eventual presence of other viral agents. In grapevine for example, HTS is powerful enough to allow for the assembly of complete genomes of the various viral species or variants infecting a sample of known or novel virus species. Rather than the serological and molecular-based methods, with their more "reductionist" view focusing on one or a few known agents, HTS-based approaches are able to give a "holistic snapshot" of the complex phytobiome of a sample of interest. In the past decade, high-throughput sequencing (HTS) has had a major impact on virus diversity studies as well as on diagnosis, providing an unbiased and more comprehensive view of the virome of a wide range of organisms.
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