Comparison of ordinary reverse transcription real-time polymerase chain reaction (qRT-PCR) with a newly developed one-step single-tube nested real-time RT-PCR (OSN-qRT-PCR) for sensitive detection of SARS-CoV-2 in wastewater.

Wastewater monitoring has proven to be an important approach to detecting and controlling the development of the SARS-CoV-2 pandemic. Various tests based on reverse transcription real-time PCR (qRT-PCR) have been developed and used for the detection of SARS-CoV-2 in wastewater samples. In this study, we attempted to increase the sensitivity of qRT-PCR by developing a one-step single-tube nested qRT-PCR assay (OSN-qRT-PCR). Two variants were developed, oriented to nucleocapsid phosphoprotein gene (N) and to spike protein gene (S), respectively. The performance of conventional qRT-PCR assays oriented to these genes with two novel OSN-qRT-PCR assays were firstly optimized using wastewater artificially contaminated with two encapsidated RNA mimic systems harboring a portion either N or S gene (ENRM and ESRM, respectively). The assays were coupled to a polyethylene glycol-based RNA precipitation/extraction method and applied to detect SARS-CoV-2 in wastewater samples from four cities in Slovakia. Both novel OSN-qRT-PCR assays demonstrated higher detection rates than the ordinary qRT-PCR counterparts. The virus levels in the analyzed wastewater samples had a high or very high relation with the numbers of clinical cases in the monitored regions. In fact, correlation with a 3-, 4-, or 5-day temporal offset was revealed. The OSN-qRT-PCR assays demonstrated robustness, mainly in samples with low viral loads.

Analysis of Hop Stunt Viroid Diversity in Grapevine (Vitis vinifera L.) in Slovakia: Coexistence of Two Particular Genetic Groups.

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the "6A" and "7A" groups based on the particular stretch of adenines at nucleotide positions 39-44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Suitability of different plant species for experimental agroinfection with Plum pox virus-based expression vector for potential production of edible vaccines.

Nine herbaceous plant species were tested for susceptibility to Plum pox virus (PPV) by Agrobacterium-mediated delivery of its infectious cDNA clone. Two of them became infected, namely spinach (local infection) and oilseed poppy (systemic infection). As a control, PPV infection was successfully established in plum seedlings following agroinfiltration, thus providing the first report of agroinfection in Prunus species. According to our results, oilseed poppy can be considered as a candidate host for the production of edible vaccines by a PPV-derived expression vector. Keywords: agroinfiltration; virus host; poppy; spinach.

Diacylglycerol Acetyltransferase Gene Isolated from Euonymus europaeus L. Altered Lipid Metabolism in Transgenic Plant towards the Production of Acetylated Triacylglycerols.

Euonymus species from the Celastraceae family are considered as a source of unusual genes modifying the oil content and fatty acid composition of vegetable oils. Due to the possession of genes encoding enzyme diacylglycerol acetyltransferase (DAcT), Euonymus plants can synthesize and accumulate acetylated triacyglycerols. The gene from Euonymus europaeus (EeDAcT) encoding the DAcT was identified, isolated, characterized, and modified for cloning and genetic transformation of plants. This gene has a unique nucleotide sequence and amino acid composition, different from orthologous genes from other Euonymus species. Nucleotide sequence of original EeDAcT gene was modified, cloned into transformation vector, and introduced into tobacco plants. Overexpression of EeDAcT gene was confirmed, and transgenic host plants produced and accumulated acetylated triacylglycerols (TAGs) in immature seeds. Individual transgenic plants showed difference in amounts of synthesized acetylTAGs and also in fatty acid composition of acetylTAGs.

Comparative Transcriptome Analysis of Two Cucumber Cultivars with Different Sensitivity to Cucumber Mosaic Virus Infection.

Cucumber mosaic virus (CMV), with extremely broad host range including both monocots and dicots around the world, belongs to most important viral crop threats. Either natural or genetically constructed sources of resistance are being intensively investigated; for this purpose, exhaustive knowledge of molecular virus-host interaction during compatible and incompatible infection is required. New technologies and computer-based "omics" on various levels contribute markedly to this topic. In this work, two cucumber cultivars with different response to CMV challenge were tested, i.e., sensitive cv. Vanda and resistant cv. Heliana. The transcriptomes were prepared from both cultivars at 18 days after CMV or mock inoculation. Subsequently, four independent comparative analyses of obtained data were performed, viz. mock- and CMV-inoculated samples within each cultivar, samples from mock-inoculated cultivars to each other and samples from virus-inoculated cultivars to each other. A detailed picture of CMV-influenced genes, as well as constitutive differences in cultivar-specific gene expression was obtained. The compatible CMV infection of cv. Vanda caused downregulation of genes involved in photosynthesis, and induction of genes connected with protein production and modification, as well as components of signaling pathways. CMV challenge caused practically no change in the transcription profile of the cv. Heliana. The main differences between constitutive transcription activity of the two cultivars relied in the expression of genes responsible for methylation, phosphorylation, cell wall organization and carbohydrate metabolism (prevailing in cv. Heliana), or chromosome condensation and glucan biosynthesis (prevailing in cv. Vanda). Involvement of several genes in the resistant cucumber phenotype was predicted; this can be after biological confirmation potentially applied in breeding programs for virus-resistant crops.

Cucumber mosaic virus resistance: Comparative proteomics of contrasting Cucumis sativus cultivars after long-term infection.

Plant viruses are a significant threat to a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant defense reactions is associated with changes in the cellular proteome to ensure virus resistance. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant host Heliana and susceptible host Vanda. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1516 reproducibly quantified proteins using a label-free approach, 133 were differentially abundant among cultivars or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Comparison of both cultivars in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection caused worse preservation of energy processes and less robust translation in the susceptible cultivar. Contrary, compatible plants had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines: A step toward durable virus resistance in cucurbits and other crops. SIGNIFICANCE: Sustainable production of crops requires an understanding of natural mechanisms of resistance/susceptibility to ubiquitous viral infections. We report original findings of comparative analysis of plant genotypes exposed to CMV. Deep discovery proteomics of resistant and susceptible cucumber cultivars, inoculated with widespread phytovirus, allowed to suggest several novel molecular targets for functional testing in plant protection strategies.

Molecular and Biological Characterisation of Turnip mosaic virus Isolates Infecting Poppy (Papaversomniferum and P. rhoeas) in Slovakia.

In recent years, the accumulated molecular data of Turnip mosaic virus (TuMV) isolates from various hosts originating from different parts of the world considerably helped to understand the genetic complexity and evolutionary history of the virus. In this work, four complete TuMV genomes (HC9, PK1, MS04, MS15) were characterised from naturally infected cultivated and wild-growing Papaver spp., hosts from which only very scarce data were available previously. Phylogenetic analyses showed the affiliation of Slovak Papaver isolates to the world-B and basal-B groups. The PK1 isolate showed a novel intra-lineage recombination pattern, further confirming the important role of recombination in the shaping of TuMV genetic diversity. Biological assays indicated that the intensity of symptoms in experimentally inoculated oilseed poppy are correlated to TuMV accumulation level in leaves. This is the first report of TuMV in poppy plants in Slovakia.

Characterization of sour cherry isolates of plum pox virus from the Volga Basin in Russia reveals a new cherry strain of the virus.

Plum pox virus (PPV) is the causal agent of sharka, the most detrimental virus disease of stone fruit trees worldwide. PPV isolates have been assigned into seven distinct strains, of which PPV-C regroups the genetically distinct isolates detected in several European countries on cherry hosts. Here, three complete and several partial genomic sequences of PPV isolates from sour cherry trees in the Volga River basin of Russia have been determined. The comparison of complete genome sequences has shown that the nucleotide identity values with other PPV isolates reached only 77.5 to 83.5%. Phylogenetic analyses clearly assigned the RU-17sc, RU-18sc, and RU-30sc isolates from cherry to a distinct cluster, most closely related to PPV-C and, to a lesser extent, PPV-W. Based on their natural infection of sour cherry trees and genomic characterization, the PPV isolates reported here represent a new strain of PPV, for which the name PPV-CR (Cherry Russia) is proposed. The unique amino acids conserved among PPV-CR and PPV-C cherry-infecting isolates (75 in total) are mostly distributed within the central part of P1, NIa, and the N terminus of the coat protein (CP), making them potential candidates for genetic determinants of the ability to infect cherry species or of adaptation to these hosts. The variability observed within 14 PPV-CR isolates analyzed in this study (0 to 2.6% nucleotide divergence in partial CP sequences) and the identification of these isolates in different localities and cultivation conditions suggest the efficient establishment and competitiveness of the PPV-CR in the environment. A specific primer pair has been developed, allowing the specific reverse-transcription polymerase chain reaction detection of PPV-CR isolates.

Evaluation of the genetic diversity of Plum pox virus 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.

The 3'-proximal part of the Plum pox virus P1 gene determinates the symptom expression in two herbaceous host plants.

Three major strains of the Plum pox virus (PPV) are the most important in Europe: PPV-D, PPV-M, and PPV-Rec. By combining the genomes of two different strains of PPV (PPV-D with PPV-Rec; PPV-D with PPV-M), 20 inter-strain chimeric infectious clones (CICPPV) were constructed. Biological properties of CICPPV were tested by inoculating them on different herbaceous host species susceptible to PPV. Four of the seven species tested, exhibited visible symptoms. In Nicotiana benthamiana all CICPPV induced systemic mosaic and leaf malformation. Pisum sativum showed a broad range of symptom severity (systemic chlorotic and necrotic lesions) but neither qualitative nor quantitative aspects of symptomatology were related to a single PPV genome locus. Nicotiana occidentalis and Nicandra physaloides proved to be suitable for symptom-based differentiation. Depending on the virus strain/chimera, N. occidentalis showed two types of symptoms: mild systemic chlorotic spots or local necrotic lesions/systemic vein necroses. N. physaloides reacted to the PPV infection either symptomless or by local necrotic lesions. Our results demonstrated that the P1/HC-pro region of the PPV genome appears to be the determinant of the symptom manifestation in these host plants. In silico analysis mapped it to the 3'-proximal part of the P1 gene.

Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup.

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.