Detection and characterisation of Plum pox virus (PPV) isolates from Eastern Slovakia revealed the presence of three main viral strains.

Plum pox virus (PPV), the agent responsible for Sharka disease, is the most important viral pathogen of stone fruit trees world-wide, having an endemic status in Slovakia. To increase knowledge of PPV diversity in Slovakia, a set of 11 isolates, originated from the eastern part of the country, was characterised. The isolates were chip-budded from their original Prunus hosts to the susceptible GF305 indicators, exhibiting the symptoms of variable severity. A genomic region encompassing the partial NIb and the hypervariable 5´terminal region of the CP gene was amplified from all 11 isolates in RT-PCR and directly sequenced. The phylogenetic analysis revealed the grouping of the 11 Slovak isolates into 3 distinct clusters, representing the PPV-M (2 isolates), D (7 isolates) and Rec strains (2 isolates). The strain affiliation of isolates was further confirmed by strain-specific RT-PCR, using which the presence of additional mixed infection by minor PPV variants was detected in 2 samples. The results further contribute to the understanding of PPV diversity in Slovakia and confirm the specificity and sensitivity of molecular approaches used for the virus strain determination

The impact of car park fire on concrete structure, Parallel computation

This study examines the influence of automobile fire in a car park on concrete parts of the structure. In 2009, a series of full-scale fire experiments in open air was conducted, including the fire in automobile interior and its influence onto a vehicle in its vicinity. We performed a set of simulations of this scenario, using the NIST FDS system, version 5.5.3. Comparison with experimental data confirmed the simulation reliability. In this paper, we use material properties of car interior materials established by our research to simulate a car fire in a small part of car park containing two burning cars and its influence on concrete ceiling and a pillar in the vicinity of the cars. We use here the calculation with 48 and more MPI processes to evaluate the ability of high performance computing to solve problems of structural fire safety

Analysis of Virome by High-Throughput Sequencing Revealed Multiple Infection and Intra-Virus Diversity in a Single Grapevine Plant

The ribosomal-depleted total RNA from white-berry grapevine (Vitis vinifera, SK933) plant showing severe chlorosis and downrolling of leaves was used for the high-throughput sequencing (HTS) analysis in order to unravel the potential contribution of the viral pathogens to the symptomatology observed. The combination of de novo assembly and mapping of ca. 1.1 millions of HTS reads enabled to identify and characterise a complex viral/viroid infection involving Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Syrah virus-1 (GSyV-1) and Hop stunt viroid (HSVd). The determined nearly complete genomes of GLRaV-2 SK933 showed its high genetic divergence from previously characterised isolates. In case of GRSPaV, two variants representing different evolutionary lineages have been identified in the plant. The results further pinpoint the complexity of grapevine viral diseases and show that mixed virus infection of grapevine is rather a rule than an exception

First report of Cucumis melo endornavirus infecting Cucurbitaceae plants in Slovakia

A symptomatic cantaloupe melon plant (Cucumis melo L.) encoded MVU2/21 grown in a garden in Velke Ulany (western Slovakia), showing pronounced chlorotic leaf spots and dwarfed growth was analysed by high-throughput sequencing (HTS). Total RNAs were isolated from apical leaves collected in August 2021. HTS was carried out on an Illumina MiSeq platform and obtained high-quality reads (∼26,4 millions, average length 131 nt) were analyzed using CLC Genomics Workbench v.10.1.1 and Geneious v.2020.2.5. BLASTn analysis of ca. 24,400 generated contigs revealed a complex viral infection involving watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV) and Cucumis melo endornavirus (CmEV, family Endornaviridae). A 15,075 nt de novo assembled contig (OL957252) corresponded to nearly complete CmEV genome with 97.9% nucleotide identity to NC_029064. Typical genome organisation and CmEV motifs, including three glucosyltransferases (Sabanadzovic et al. 2016), were found in MVU2/21 polyprotein. The MVU2/21 total RNAs were subsequently subjected to RT-PCR using primers reported by Zeng et al. (2020) as well as two newly designed primer pairs: CmE_14443F (5´-TATGCGGTTGACTGGACAGG-3´)/CmE_14931R (5´-TTATGAGCCACAGCGGTCAC-3´) and CmEV_4145F (5´-CTTGCCTTGAAGGTAGATCG-3´)/ CmEV_4634R (5´-GATTGTCCGCACCGTATAAC-3´). Sanger sequencing of the specific 489, 490 and 710 bp PCR products unambiguously confirmed the HTS data. In order to analyse the potential presence of CmEV in Slovakia, additional leaf samples from melons, cucumbers and squashes from four different localities were tested by RT-PCR using the above primers. Five out of 40 samples collected from different locations and hosts, i.e. melon (MVU1/21), cucumber (UVU1/21, UVU2/21), squash (TPE 2/21) and patison (CPTV6) tested positive for CmEV. Partial genomic sequences of all Slovak isolates amplified using CmEV_4145F/4634R primers (OL957253-OL957257) shared high nucleotide identity to MVU2/21 (97.2–100%). CmEV was previously reported outside Europe (Sabanadzovic et al. 2016; da Costa et al. 2019; Zeng et al. 2020). Our work thus extents data on CmEV geographical distribution and host range

A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll Associated Virus 1

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer

The Expanding Menagerie of Prunus-Infecting Luteoviruses

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.This study was funded by the European Union through the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-60 ITN) project “INEXTVIR” (grant agreement number 813542). The ChLVA research part was financed by the Academy of Sciences of the Czech Republic (RVO60077344). The plant indexing biological tests conducted by CTIFL were funded by INTERFEL (fresh fruit and vegetable interprofessional association). D. Safarova and M. Navratil received support from the Ministry of Agriculture of the Czech Republic, National Agency for Agricultural Research (project QK1920124). M. Glasa and D. Mihálik received support from the Slovak Research & Development Agency (project APVV-18-0005)Peer reviewe

Plum Pox Virus Genome-Based Vector Enables the Expression of Different Heterologous Polypeptides in Nicotiana benthamiana Plants

Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system

Aphid transmission of natural recombinant plum pox virus isolates to different prunus ssp-a contribution for understanding the epidemiology of an atypical PPV

UMR BGPI Equipe 6International audiencePlum pox virus (PPV), a member of the Potyvirus genus, is distributed worldwide and is the causal agent of the economically important sharka disease of stone-fruit trees. Recently, natural recombinant M/D isolates were found to occur in Slovakia and several European countries. Surprisingly, all the recombinant isolates identified to date showed a very close molecular relationship and shared the same position of a recombination breakpoint situated in the C terminus of the NIb gene. To verify the ability of recombinant PPV to be naturally transmitted by aphids and thus to evaluate their possible epidemiological impact, experimental transmission of temporally and geographically distant recombinant PPV isolates to different Prunus spp. (P. domestica ‘Julior’, P. armeniaca ‘Manicot’, P. persica ‘GF305’ and ‘Montclar’) was carried out using a clonal culture of Myzus persicae Sulzer under controlled conditions using a technique involving a controlled acquisition access period. The results confirmed that all the recombinant PPV isolates were transmitted by aphids; however, the transmission occurred at different rates

Evaluation of the genetic diversity of <em>Plum pox virus</em> in a single plum tree.

Genetic diversity of Plum pox virus (PPV) and its distribution within a single perennial woody host (plum, Prunus domestica) has been evaluated. A plum tree was triply infected by chip-budding with PPV-M, PPV-D and PPV-Rec isolates in 2003 and left to develop untreated under open field conditions. In September 2010 leaf and fruit samples were collected from different parts of the tree canopy. A 745-bp NIb-CP fragment of PPV genome, containing the hypervariable region encoding the CP N-terminal end was amplified by RT-PCR from each sample and directly sequenced to determine the dominant sequence. In parallel, the PCR products were cloned and a total of 105 individual clones were sequenced. Sequence analysis revealed that after 7 years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and PPV-D) had been displaced. Despite the fact that the analysis targeted a relatively short portion of the genome, a substantial amount of intra-isolate variability was observed for PPV-M. A total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely dominant. However, no clear-cut structuration of the viral population by the tree architecture could be highlighted although the results obtained suggest the possibility of intra-leaf/fruit differentiation of the viral population. Comparison of the consensus sequence with the original source isolate showed no difference, suggesting within-plant stability of this original isolate under open field conditions