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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.

The Erysimum latent virus (ELV), a tymovirus, was first isolated from several wild and cultivated brassicas in Germany. Its virions were shown to be serologically distinct from those of the turnip yellow mosaic virus (TYMV), which is also found in wild and cultivated plants in several European countries but also in other parts of the world. TYMV and ELV were among the first plant viruses to have had their genomes sequenced, and when other tymovirus genomes were sequenced, it was found that, in phylogenies, ELV is probably the basal outlier to all other tymoviruses. Here, we report the near-complete genomic sequence of another isolate of ELV from Czechia. This isolate was found in 1990 in Sisymbrium altissimum plants showing mosaic symptoms. It was detected using ELISA tests and electron microscopy. We have now sequenced the full coding sequence of this isolate using contemporary high throughput methods and found that the German and Czech isolates of ELV are closely related and are of the same virus species.

This study focused on the viruses of the Tymoviridae family that infect grapevines in the Czech Republic. Complete sequences of GFkV (grapevine fleck virus) and GRGV (grapevine red globe virus) from the genus Maculavirus and GRVFV (grapevine rupestris vein feathering virus) and GSyV-1 (grapevine Syrah virus 1) from the genus Marafivirus 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 Tymoviridae 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 Marafivirus, was reported for the first time in the Czech Republic.

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.

A field trial with plum cv. HoneySweet was established in 2003 in an experimental plot near Crop Research Institute in Prague-Ruzyně, Czech Republic, on the basis of a permission issued by the Ministry of the Environment of the Czech Republic. In addition to the evaluation of resistance of plum cv. HoneySweet to Plum pox virus, the field trial was used to evaluate gene flow of the inserted transgene. Sampling of blackthorn and myrobalan trees outside the field test site occurred at distances ranging from 544 m to 845 m from the test site and showed no gene flow, testing both plants and seeds collected from blackthorns and myrobalans. Similarly, seeds from plums cv. Jojo growing directly at the field test place did not show any presence of the transgene after seven years of evaluation.

Resistance to Plum pox virus (PPV) in transgenic Prunus domestica L., clone C5 (cv. HoneySweet) was evaluated in a regulated field in the Czech Republic for fifteen years (2002-2016). PPV mild symptoms appeared in C5 trees only in several leaves situated close to the point of inoculum grafting up to 2010. No symptoms of PPV were observed in the years 2011-2013 and results of ELISA and RT-PCR detection tests were negative. In the twelfth year (2013), there was a severe unusual natural attack of plum trees by Monilinia sp. This Monilinia sp. attack occurred only one time - in 2013. There was no Monilinia sp. infection in 2002-2012 and in 2014-2016. Mild PPV symptoms reappeared in several leaves of transgenic plum trees in the next two years (2014-2015) and the presence of PPV was proved by DAS-ELISA and confirmed by RT-PCR.

Sharka caused by Plum pox virus (PPV) is the most harmful disease of stone fruits in Europe and elsewhere in the world. There is no highly PPV resistant cultivar of plum. Biotech approach has led to the development of resistance through genetic engineering. In this study, we evaluated a transgenic plum Prunus domestica L., clone C5 (cv. HoneySweet), where the PPV resistance is based on RNA interference (RNAi). Resistance in C5 plums has been evaluated for PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) in a regulated field trial in the Czech Republic for 12 years (2002–2013). Even under high and permanent infection pressure introduced through graft inoculation of the viruses, PPV has been detected in C5 trees only in several leaves situated close to the point of inoculum grafting in the first nine years. Mild symptoms of PPV disappeared year by year. No PPV symptoms were observed in the last three years and results of ELISA detection were negative. Similar results were obtained, when RT-PCR was used for PPV detection. No natural infection of PPV by aphids was recorded in C5 plums, even if PPV infected plants were growing in the same experimental field in close vicinity. Co-infections of PPV with PDV and/or ACLSV had practically no influence on stability of resistance in C5 trees. Twelve years of field testing in the Czech Republic clearly demonstrated the high level of resistance of C5 either to PPV infection by graft inoculation or natural infection through aphid vectors.

Nine different visual evaluations of Plum pox virus (PPV) presence were carried out in four nurseries during 2012-2015. Results of visual evaluation were verified by ELISA. The presence of PPV was confirmed by ELISA in all the trees showing PPV symptoms. PPV has never been detected by ELISA in trees without PPV symptoms on leaves. A very low occurrence of PPV was proved in plums in all the four nurseries. There were two cases of higher occurrence of PPV in plum cultivars, where PPV originated from infected grafts. The PPV occurrence was 0.126 and 1.59%. The average occurrence of PPV in plums was 0.075%, 93 trees out of 123 630 inspected were PPV-infected. All the PPV-infected trees were destroyed immediately. No new PPV infection was proved later in season (August-September). In comparison with the situation in the 60s of the last century, the PPV occurrence in plums was 2.48% in one nursery in 1963 and there were nurseries with PPV occurrence exceeding 5%. All growing plants were destroyed in this case. The occurrence of PPV in the Czech fruit nurseries today is more than hundred times lower in comparison with the situation fifty years ago. The incidental occurrence of PPV in nurseries cannot be excluded in countries and areas with the endemic presence of PPV (in the Czech Republic and in most European countries). Visual inspection of PPV symptoms in nurseries confirmed by ELISA testing is sufficient. Infected plants must be removed immediately.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

Ten-year results of the practical immunity investigation of apricot cv. Harlayne are presented. Two-year-old trees of cv. Harlayne were inoculated by chip-budding with six different strains and isolates of Plum pox virus (PPV). PPV inoculated trees grew in the field and were evaluated from 2001 to 2011. No PPV symptoms appeared in the leaves of cv. Harlayne within ten years (2002-2011), and within eight years (2004-2011) in the fruits and stones. None of the six isolates of three different PPV strains was detected in the leaves and fruits by ELISA. Suckers of the rootstock Prunus myrobalana developed around cv. Harlayne trees in 2005-2011 were symptomless and ELISA was negative within seven years. New trees of cv. Harlayne obtained from tested trees by budding on PPV susceptible apricot rootstock MVA-2 in 2007 were PPV free from 2008 through 2011. The presence of PPV was proved by ELISA neither in leaves of cv. Harlayne nor in rootstock MVA-2.

The distribution of Plum pox virus (PPV) strains, PPV-D, PPV-M and PPV-Rec, was investigated in the Czech Republic in 2005-2008. Fifty-two to ninety-four samples of flowers or leaves of plum, myrobalan and blackthorn trees from different regions were tested in individual years. The presence of PPV was detected by DAS-ELISA with serotype-specific polyclonal antibodies. PPV-M was proved by DASI-ELISA with serotype-specific monoclonal antibodies; PPV-D, PPV-M and PPV-Rec were detected by RT-PCR in leaf samples from PPV infected trees. The presence of PPV-D ranged from 94.6% to 100%, the presence of PPV-M from 0.0% to 2.3% and the presence of PPV-Rec from 0.0% to 3.1% during 2005-2008. More than 95% of analysed samples of PPV were infected with PPV-D and less than 2.5% of analysed samples of PPV were infected with PPV-M or PPV-Rec. The presence of PPV-C was not proved in sweet cherry and sour cherry trees. The presence of PPV-EA was not proved in apricot trees.

An isolate of Grapevine virus A (GVA) from the Czech Republic was obtained from the grapevine cultivar Müller Thurgau. Symptoms of GVA - Kober stem grooving disease were not observed in the infected grapevines (which had been grafted onto Kober 5BB rootstock). A partial genomic sequence of the GVA isolate, 1523 nucleotides long, was obtained. The sequence completely covers the genes for both a movement and a coat protein. Compared to the GVA sequences available in databases, the nucleotide identity reached 84%. The amino acid identity in the movement protein reached 88%, and 98% in the coat protein.

Electron microscopy was used to detect the presence of virus particles or inclusions in growth tips and parenchymatic cells of leaves of plum, apricot and peach trees artificially infected with Plum pox virus (PPV). Typical pinwheels were found in ultrathin sections of leaves of PPV infected plums, apricots and peaches. Filamentous particles or their aggregates approximately 750 nm long were found in ultrathin sections of growth tips of plum, apricot, and peach shoots with a diameter of 0.5 mm. Pinwheels were never present in embryonic cells. No virus particles were found in ultrathin sections of growth tips of PPV infected plum, apricot and peach shoots of 0.2 mm in diameter. Embryonic cells of growth tips up to 0.2 mm in diameter are PPV free. PPV particles are present in growth tips at a distance 0.2-0.5 mm from the top; the virus is probably multiplied in this part of the growth tips.

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.