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.
Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based ...on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prunus necrotic ringspot virus (PNRSV) and cherry virus A (CVA) are two viruses that mainly infect plants of the genus Prunus. Full-length sequences of these two viruses, collected in the Czech ...Republic from Prunus cerasifera plants, were obtained via HTS sequencing. Phylogenetic analyses based on the NJ method and Splitstree tools showed that the Czech PNRSV isolate (ON088600-ON088602) is a divergent isolate from other molecular groups, sharing less than 97% pairwise nucleotide identity with members of other groups. The Czech CVA isolate (ON088603) belonged to molecular subgroup III-2, clustered with isolates from non-cherry hosts, and shared the highest pairwise nucleotide identity (99.7%) with an isolate of Australian origin.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This study focused on the viruses of the
family that infect grapevines in the Czech Republic. Complete sequences of GFkV (grapevine fleck virus) and GRGV (grapevine red globe virus) from the genus
...and GRVFV (grapevine rupestris vein feathering virus) and GSyV-1 (grapevine Syrah virus 1) from the genus
were obtained using high-throughput sequencing of small RNAs and total RNAs. Mixed infections with these viruses were observed, as well as several variants of these viruses in the same plant. Phylogenetic analysis showed the position of the newly obtained virus isolates within the
family. Recombinant analysis provided evidence of single and multiple intraspecific recombinations in GRGV, GSyV-1, and GRVFV. Additionally, GAMaV, a grapevine virus from the genus
, was reported for the first time in the Czech Republic.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred ...growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.
A multiplex RT-PCR reaction was developed and validated for the simultaneous detection of two viruses and two viroids: grapevine fleck virus, grapevine Pinot gris virus, grapevine yellow speckle ...viroid 1, and hop stunt viroid. The multiplex reaction specificity was verified on a set of 210 grapevines from the Czech Republic. The results showed the occurrence of hop stunt viroid in 94.3% of the examined plants, while incidence of the other pathogens was lower: grapevine Pinot gris virus (64.8%), grapevine yellow speckle viroid 1 (52.4%), and grapevine fleck virus (15.2%).
Abstract
Watermelon mosaic virus (WMV) is a potyvirus and a member of the bean common mosaic virus (BCMV) lineage. It is one of the most economically important viral pathogens of cucurbits worldwide ...and was first reported in the Czech Republic in 2011 from serological surveys (2005–2011). In this study, we confirmed this identification by determining the complete coding regions of five Czech WMV isolates using high‐throughput sequencing and Sanger sequencing (MW188031; OP585149–OP585152), together with the coat protein (CP) genes of 26 additional isolates. Phylogenies were made from these and more than 128 genomes or 128 CP genes from GenBank. They showed that the Czech isolates were most closely related to other European isolates, but, surprisingly, 96.2% of the genomes were recombinant. The nonrecombinant sequences mostly came from basal isolates, all originating from China, and some from unusual hosts (
Ailanthus altissima
,
Alcea rosea
and
Panax ginseng
). The complete WMV genomes form three phylogenetic clades, two of them small and basal, and the third includes all other isolates. Comparative dating suggests that the basal Chinese isolates are descendants of a potyvirus population infecting various dicotyledonous plant species in China at least 2000 years ago. WMV became a crop pathogen around 1000 years ago, a few years after watermelon was taken to northern China and first grown as a crop during the Five Dynasties (907–960
ce
).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•Small RNA sequencing was used to evaluate healthy status of grapevines.•Repeated Ribavirin treatment successfully removed viruses present in the plants.•Viroids remained in the plants despite the ...Ribavirin treatment.•Virus sanitation was still durable three years after the Ribavirin treatment.
This study describes the application of high-throughput sequencing of small RNA analysis of the efficacy of using Ribavirin to eliminate Grapevine leafroll-associated virus 1, Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus from Vitis vinifera cv. Riesling. The original plant used for sanitation by Ribavirin treatment was one naturally infected with all the viruses mentioned above as confirmed by RT-PCR. A tissue cultures of the plant were established and plantlets obtained were sanitized using Ribavirin. Three years after sanitation, a small RNA sequencing method for virus detection, targeting 21, 22 and 24 nt-long viral small RNAs (vsRNAs), was used to analyze both the mother plant and the sanitized plants. The results showed that the mother plant was infected by the three mentioned viruses and additionally by two viroids - Hop stunt viroid and Grapevine yellow speckle viroid 1. After Ribavirin treatment, the plants contained only the two viroids, with the complete elimination of all the viruses previously present.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Filamentous virus particles were found in Pleione orchids using electron microscopy.•High-throughput sequencing was used to identify and characterise a distinct potyvirus.•The name Pleione flower ...breaking virus was suggested for the virus.•The virus was successfully transmitted by aphids Myzus persicae Sulz.
Plants of the genus Pleione, originating from hobby growers in the Netherlands and in the Czech Republic, were conspicuous for viral infection, showing symptoms of leaf mosaic or flower breaking. Using Sanger and high throughput sequencing, the full genome sequence of a novel potyvirus was obtained from sequencing data. The genome sequence was annotated and compared to the genome of other potyviruses. The virus was experimentally transmitted by aphids into Pleione and Chenopodium quinoa plants. The name Pleione flower breaking virus (PlFBV) was suggested for the new virus. The presence of the virus was confirmed using RT-PCR, with newly designed primers targeting this new species. The incidence of the virus was contrasted between both countries and might have been influenced by the growth conditions and the exposure of the plants to aphids.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
1 Institute of Virology, Department of Plant Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 84505 Bratislava, Slovakia
2 Agricultural Biotechnology Center, Environmental Biosafety ...Institute, PO Box 411, H-2101 Godollo, Hungary
3 Research Institute of Crop Production, Drnovská 507, 161 06 Prague Ruzyn , Czech Republic
4 Equipe Epidémiologie Virus/Vecteurs, UMR BGPI CIRAD TA 41/K, Campus International de Baillarguet, 34398 Montpellier, France
5 UMR GDPP, INRA et Université Bordeaux 2, IBVM, Centre INRA de Bordeaux, BP 81, 33883 Villenave d'Ornon Cedex, France
Correspondence Miroslav Glasa virumig{at}savba.sk
Natural recombinant Plum pox virus (PPV) isolates were detected in Albania, Bulgaria, Czech Republic, Germany, Hungary and Slovakia. Despite different geographical origins and dates of isolation, all the recombinant isolates were closely related at the molecular level and shared the same recombination breakpoint as well as a typical signature in their N-terminal coat protein sequence, suggesting a common origin. Biological assays with four recombinant isolates demonstrated their capacity to be aphid-transmitted to various Prunus hosts. One of these isolates had a threonine-to-isoleucine mutation in the conserved PTK motif of its HC-Pro and showed a drastically decreased, although not abolished, aphid transmissibility. The complete genome sequence of one of the recombinant isolates, BOR-3, was determined, as well as some partial sequences in the HC-Pro and P3 genes for additional natural recombinant isolates. Analysis of the phylogenetic relationships between the recombinant isolates and other sequenced PPV isolates confirmed that the recombinant isolates form a phylogenetically homogeneous lineage. In addition, this analysis revealed an ancient recombination event between the PPV-D and M subgroups, with a recombination breakpoint located in the P3 gene. Taken together, these results indicate that recombinant isolates represent an evolutionarily successful, homogeneous group of isolates with a common history and unique founding recombination event. The name PPV-Rec is proposed for this coherent ensemble of isolates.
The GenBank accession numbers of the nucleotide sequences reported in this paper are AY028309 , AY553368 AY553377 , AY324837 AY324848 , AJ566344 AJ566346 , AJ620686 AJ620687 .
Details of PCR primers and a multiple alignment of PPV sequences are available as supplementary material in JGV Online.
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