Detection and molecular characterization of Slovak tomato isolates belonging to two recombinant strains of potato virus Y

Sihelská, Nina; Predajňa, Lukáš; Nagyová, A; Šoltýs, Katarína; Budiš, Jaroslav; Gubiš, Jozef; Mrkvová, Michaela; Kraic, Ján; Mihálik, Daniel; Glasa, Miroslav

doi:10.4149/av_2016_04_347

Cited by 8 publications

(4 citation statements)

References 26 publications

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“…In all these cases, a single PVY variant was unambiguously identified, belonging to the PVY-N-Wi, PVY-NTNa, or PVY-NTNb strains. These results complement a previous more limited work [34], showing the presence of recombinant PVY strains in Slovakia based on RT-PCR targeting various recombination junction (RJ) sites.…”

Section: Discussionsupporting

confidence: 88%

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Glasa

Hančinský

Šoltýs

et al. 2021

Plants

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In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

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Section: Discussionsupporting

confidence: 88%

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Glasa

Hančinský

Šoltýs

et al. 2021

Plants

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“…Based on the literature, for total RNA extracts lacking ribosomal RNA depletion, the percentage of viral reads for HTS is reported to range from 0.5% to 2% [17, 38, 39]. By removing ribosomal RNA prior to library preparation, between 0.02% and 6% virus-specific reads can be obtained [9, 11, 40, 41]. In our study, between 0.05% and 11% virus-specific reads could be obtained (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences

2019

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Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus .

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“…The cDNA was then column-purified with the DNA Clean & Concentrator ™ -5 – DNA kit (Zymo Research, Irvine, USA) and quantified with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The sample was then processed with the transposon-based chemistry library preparation kit (Nextera XT, Illumina) as previously described ( Sihelská et al, 2016 ). The fragment size structure of the DNA library was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).…”

Section: Determination Of the Tomv Sl-1 Full Length Genome Sequence Bmentioning

confidence: 99%

Experimental Infection of Different Tomato Genotypes with Tomato mosaic virus Led to a Low Viral Population Heterogeneity in the Capsid Protein Encoding Region

Sihelská

Vozárová

Predajňa

et al. 2017

The Plant Pathology Journal

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The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semi-quantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08–0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection.

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Detection and molecular characterization of Slovak tomato isolates belonging to two recombinant strains of potato virus Y

Cited by 8 publications

References 26 publications

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences

Experimental Infection of Different Tomato Genotypes with Tomato mosaic virus Led to a Low Viral Population Heterogeneity in the Capsid Protein Encoding Region

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