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BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.
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Coinfecção , Euphorbia , Ácidos Nucleicos , Vírus de Plantas , Potyviridae , Vírus de RNA , Inosina Trifosfatase , Filogenia , Vírus de RNA/genética , Nucleotídeos/genética , Potyviridae/genética , Vírus de Plantas/genética , Plantas/genética , RNA Viral/genética , Genoma ViralRESUMO
Despite the fact that Cladosporium sp. are ubiquitous fungi, their viromes have been little studied. By analysing a collection of Cladosporium fungi, two new partitiviruses named Cladosporium cladosporioides partitivirus 1 (CcPV1) and Cladosporium cladosporioides partitivirus 2 (CcPV2) co-infecting a strain of Cladosporium cladosporioides were identified. Their complete genome consists of two monocistronic dsRNA segments (RNA1 and RNA2) with a high percentage of pairwise identity on 5' and 3' end. The RNA directed RNA polymerase (RdRp) of both viruses and the capsid protein (CP) of CcPV1 display the classic characteristics required for their assignment to the Gammapartitivirus genus. In contrast, CcPV2 RNA2 encodes for a 41 KDa CP that is unusually smaller when aligned to CPs of other viruses classified in this genus. The structural role of this protein is confirmed by electrophoresis on acrylamide gel of purified viral particles. Despite the low percentage of identity between the capsid proteins of CcPV1 and CcPV2, their three-dimensional structures predicted by AlphaFold2 show strong similarities and confirm functional proximity. Fifteen similar viral sequences of unknown function were annotated using the CcPV2 CP sequence. The phylogeny of the CP was highly consistent with the phylogeny of their corresponding RdRp, supporting the organization of Gammapartitiviruses into three distinct clades despite stretching the current demarcation criteria. It is proposed that a new subgenus be created within the genus Gammapartitivirus for this new group.
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Micovírus , Vírus de RNA , Cladosporium/genética , Micovírus/genética , Vírus de RNA/genética , Proteínas do Capsídeo/genética , Fungos , RNA Polimerase Dependente de RNA/genéticaRESUMO
Leaves of hollyhock (Alcea rosea) exhibiting vein chlorosis and yellow mosaic symptoms were collected at public sites in Lausanne and Nyon, two cities of western Switzerland. Diagnostic methods untangled in samples from both sites the mixed infections of a novel isometric virus, tentatively named "Alcea yellow mosaic virus" (AYMV) with the carlavirus Gaillardia latent virus. A new potyvirus was also identified in samples from Nyon. A combination of Illumina, Nanopore and Sanger sequencing was necessary to assemble the full-length genome of AYMV, revealing an exceptionally high cytidine content and other features typically associated with members of the genus Tymovirus. The host range of AYMV was found to be restricted to mallows, including ornamentals as well as economically important plants. Phylogenetic analyses further showed that AYMV belongs to a Tymovirus subclade that also gathers the other mallow-infecting members. The virus was readily transmitted by sap inoculation, and the weevil species Aspidapion radiolus was evidenced as a vector. Transmission assays using another weevil or other insect species did not succeed, and seed transmission was not observed.
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Coinfecção , Malvaceae , Vírus do Mosaico , Tymovirus , Gorgulhos , Animais , Tymovirus/genética , Filogenia , Doenças das PlantasRESUMO
Every year, Agroscope examines nearly 300,000 tubers for the presence of viruses, as regulated for the certification of seed potatoes intended for Swiss growers. Since 2016, this examination has been performed via RT-qPCR on dormant tubers directly after harvest. This method offers fast results and eliminates the need for the use of Rindite, which is a toxic and polluting gaseous compound previously used in Switzerland to break the dormancy of seed tubers. The implementation of this molecular analytical method for the routine diagnosis of regulated viruses makes it possible to conduct additional analyses via Illumina sequencing to assess the conformity of the primers and probes used with the sequences of the different viral isolates. This form of quality control in routine diagnosis is a source of information that can answer more fundamental scientific questions related to the epidemiology of viral strains related to certification. The datasets produced in this framework can also be used to explore the diversity of rare or unknown virus species in potato crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11540-021-09491-3.
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We report the genome sequence of a putative new foveavirus infecting non-cultivated Vitis vinifera, tentatively named "grapevine foveavirus A" (GFVA). This virus was identified by high-throughput sequencing analysis of a European wild Vitis collected in Switzerland. Phylogenetic analysis revealed that this virus clustered with known grapevine virus T (GVT) isolates but was clearly distinct from any of them. If considering the International Committee of Taxonomy of Viruses (ICTV)-suggested foveavirus species demarcation criterion based on sequence similarity in the replicase gene/protein, this virus should be considered a member of a new species closely related to GVT. On the other hand, comparison of capsid gene/protein sequences using the same criteria indicates that GFVA is at the border of species demarcation. Whether this virus represents a highly divergent GVT isolate or a member of a distinct but closely related species is discussed.
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Flexiviridae/classificação , Genoma Viral , Filogenia , Vitis/virologia , Flexiviridae/isolamento & purificação , Variação Genética , Doenças das Plantas/virologia , SuíçaAssuntos
DNA Bacteriano , Paenibacillus , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Abelhas/microbiologia , Animais , Paenibacillus/isolamento & purificação , Paenibacillus/classificação , Paenibacillus/genética , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Ácidos Graxos , Composição de BasesRESUMO
In plants, RNA silencing-based antiviral defense generates viral small RNAs (sRNAs) faithfully representing the viral genomes. We employed sRNA sequencing and bioinformatics (sRNA-omics) to characterize antiviral defense and to reconstruct the full genomic sequences and their variants in the evolving viral quasispecies in cultivated solanaceous plants carrying mixed infections. In naturally infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo-assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs, which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (genus Potexvirus) was tested for protection against strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically nontransmissible Southern tomato virus (genus Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA-generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes.
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Genoma Viral , Doenças das Plantas/virologia , Potexvirus/genética , Potyvirus/genética , Solanum , Coinfecção , Potexvirus/isolamento & purificação , Potyvirus/isolamento & purificação , Interferência de RNA , RNA Viral , Solanum/virologiaRESUMO
Grapevine red blotch virus (GRBV) is a recently described virus that infects grapevine. Little information is available on the possible occurrence and distribution outside North America. Therefore, we surveyed commercial vineyards from the three major grape-growing regions in Switzerland to determine the presence or absence of GRBV. In total, 3,062 vines were analyzed by polymerase chain reaction. None of the vines tested positive for GRBV, suggesting the absence of GRBV from Swiss vineyards. We also investigated whether GRBV was present in 653 grapevine accessions in the Agroscope grapevine virus collection at Nyon, including dominantly Swiss (457) but also international accessions. Only six referential accessions were infected by GRBV, all originating from the United States, whereas all others from 10 European and 8 non-European origins tested negative. High-throughput sequencing analysis of Zinfandel A2V13, in the collection since 1985, confirmed close similarity of GRBV isolate Z_A2V13 to American isolates according to genomes deposited in GenBank. Because the Zinfandel A2V13 reference was also maintained grafted on the leafroll virus indicator Vitis vinifera 'Gamay', we evaluated the effect of GRBV on viticultural performance over a 3-year period. Our results showed clear detrimental effects of GRBV on grapevine physiology (vine vigor, leaf chlorophyll content, and gas exchange) and fruit quality. These findings underscore the importance of implementation of GRBV testing worldwide in certification and quarantine programs to prevent the dissemination of this virus.
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Geminiviridae/fisiologia , Doenças das Plantas/virologia , Vitis/virologia , Frutas/virologia , Folhas de Planta/virologia , Suíça , Vitis/fisiologiaRESUMO
The ability of fungi isolated from nails of patients suffering from onychomycosis to induce de novo production of bioactive compounds in co-culture was examined. Comparison between the metabolite profiles produced by Sarocladium strictum, by Fusarium oxysporum, and by these two species in co-culture revealed de novo induction of fusaric acid based on HRMS. Structure confirmation of this toxin, using sensitive microflow NMR, required only three 9-cm Petri dishes of fungal culture. A targeted metabolomics study based on UHPLC-HRMS confirmed that the production of fusaric acid was strain-dependent. Furthermore, the detected toxin levels suggested that onychomycosis-associated fungal strains of the F. oxysporum and F. fujikuroi species complexes are much more frequently producing fusaric acid, and in higher amount, than strains of the F. solani species complex. Fusarium strains producing no significant amounts of this compound in pure culture, were shown to de novo produce that compound when grown in co-culture. The role of fusaric acid in fungal virulence and defense is discussed.
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Técnicas de Cocultura , Ácido Fusárico/biossíntese , Fusarium/metabolismo , Onicomicose/microbiologia , Meios de Cultura/química , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
We report the characterization of a novel tri-segmented RNA virus infecting Mercurialis annua, a common crop weed and model species in plant science. The virus, named "Mercurialis latent virus" (MeLaV) was first identified in a mixed infection with the recently described Mercurialis orthotospovirus 1 (MerV1) on symptomatic plants grown in glasshouses in Lausanne (Switzerland). Both viruses were found to be transmitted by Thrips tabaci, which presumably help the inoculation of infected pollen in the case of MeLaV. Complete genome sequencing of the latter revealed a typical ilarviral architecture and close phylogenetic relationship with members of the Ilarvirus subgroup 1. Surprisingly, a short portion of MeLaV replicase was found to be identical to the partial sequence of grapevine angular mosaic virus (GAMV) reported in Greece in the early 1990s. However, we have compiled data that challenge the involvement of GAMV in angular mosaic of grapevine, and we propose alternative causal agents for this disorder. In parallel, three highly-conserved MeLaV isolates were identified in symptomatic leaf samples in The Netherlands, including a herbarium sample collected in 1991. The virus was also traced in diverse RNA sequencing datasets from 2013 to 2020, corresponding to transcriptomic analyses of M. annua and other plant species from five European countries, as well as metaviromics analyses of bees in Belgium. Additional hosts are thus expected for MeLaV, yet we argue that infected pollen grains have likely contaminated several sequencing datasets and may have caused the initial characterization of MeLaV as GAMV.
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Genoma Viral , Ilarvirus , Filogenia , Doenças das Plantas , Pólen , Vitis , Vitis/virologia , Doenças das Plantas/virologia , Pólen/virologia , Ilarvirus/genética , Ilarvirus/isolamento & purificação , Ilarvirus/classificação , Animais , RNA Viral/genética , Sequenciamento Completo do Genoma , Tisanópteros/virologiaRESUMO
In the original publication [...].
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The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4â³-hydroxysulfoxy-2,2â³-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.
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Hypocreales/crescimento & desenvolvimento , Trichophyton/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Técnicas de Cocultura , Meios de Cultura , Hypocreales/química , Hypocreales/genética , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Trichophyton/química , Trichophyton/genéticaRESUMO
Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.
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Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Humanos , Polimorfismo de Nucleotídeo Único/genética , Genoma Viral/genética , Biologia Computacional , ConhecimentoRESUMO
High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.
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Members of the genus 'Candidatus Phytoplasma' are obligate intracellular bacteria restricted to phloem sieve elements and are able to colonize several tissues and the hemolymph in their insect vectors. The current unfeasibility of axenic culture and the low complexity of genomic sequences are obstacles in assembling complete chromosomes. Here, a method combining pathogen DNA enrichment from infected insects and dual deep-sequencing technologies was used to obtain the complete genome of a phytoplasma causing Grapevine Flavescence dorée. The de novo assembly generated a circular chromosome of 654,223 bp containing 506 protein-coding genes. Quality assessment of the draft showed a high degree of completeness. Comparative analysis with other phytoplasmas revealed the absence of potential mobile units and a reduced amount of putative phage-derived segments, suggesting a low genome plasticity. Phylogenetic analyses identified Candidatus Phytoplasma ziziphi as the closest fully sequenced relative. The "Flavescence dorée" phytoplasma strain CH genome also encoded for several putative effector proteins potentially playing a role in pathogen virulence. The availability of this genome provides the basis for the study of the pathogenicity mechanisms and evolution of the Flavescence dorée phytoplasma.
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Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant.
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Closteroviridae , Vitis , Closteroviridae/genética , Flexiviridae , Doenças das PlantasRESUMO
Massive outbreaks of virus yellows (VY) and syndrome "basses richesses" (SBR) are thought to be responsible for the major loss of sugar beet yields in 2020 in western cantons of Switzerland. Typical yellowing symptoms were visible during field inspections, and control measures were reportedly ineffective or even absent. Both diseases induce yellowing but have distinct etiologies; while VY is caused by aphid-transmitted RNA viruses, SBR is caused by the cixiid-transmitted γ-proteobacterium Candidatus Arsenophonus phytopathogenicus. To clarify the situation, samples from diseased plants across the country were screened for the causal agents of VY and SBR at the end of the season. Beet yellows virus (BYV) and Beet chlorosis virus (BChV) showed high incidence nationwide, and were frequently found together in SBR-infected fields in the West. Beet mild yellowing virus (BMYV) was detected in two sites in the West, while there was no detection of Beet western yellows virus or Beet mosaic virus. The nucleotide diversity of the detected viruses was then investigated using classic and high-throughput sequencing. For both diseases, outbreaks were analyzed in light of monitoring of the respective vectors, and symptoms were reproduced in greenhouse conditions by means of insect-mediated inoculations. Novel quantification tools were designed for BYV, BChV and Ca. A. phytopathogenicus, leading to the identification of specific tissues tropism for these pathogens.
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Flavescence dorée (FD) is a grapevine disease caused by 'Candidatus Phytoplasma vitis' (FDp), which is epidemically transmitted by the Nearctic leafhopper Scaphoideus titanus. In this study, we applied dendrochronological techniques to analyse the response to FDp infections in terms of wood ring widths and anatomical structures of the xylem and phloem tissues of the trunk of the susceptible grapevine cultivar 'Chardonnay.' As a rule, grapevines are susceptible to water shortage and reduce their growth in diameter in case of summer drought. In the season of the external expression of FD symptoms, however, the ring width reductions are extreme and supersede any drought-induced effects. In addition, the anatomy of the phloem tissue in the year of the FD symptom expression appears heavily disarranged. Moreover, in the most suffering individuals, the xylem formation remains incomplete and mostly limited to the early wood tissue. In conclusion, even though the FD phytoplasma does not inhabit and replicate inside the xylem tissue, our results confirm existing indirect inhibiting effects on the ring growth and the xylem tissue formation in FDp-infected grapevines.
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Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a â¼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.
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High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.