1. Assessment of different experimental setups to determineviral filtration efficiency of face masksArijana Filipić, Katja Fric, Maja Ravnikar, Polona Kogovšek, 2022, izvirni znanstveni članek Povzetek: Abstract
As a result of the COVID-19 pandemic, many new materials and masks came onto the market. To determine their suitability, several standards specify which properties to test, including bacterial filtration efficiency (BFE), while none describe how to determine viral filtration efficiency (VFE), a property that is particularly important in times of pandemic. Therefore, we focused our research on evaluating the suitability and efficiency of different systems for determining VFE. Here, we evaluated the VFE of 6 mask types (e.g., a surgical mask, a respirator, material for mask production, and cloth masks) with different filtration efficiencies in four experimental setups and compared the results with BFE results. The study included 17 BFE and 22 VFE experiments with 73 and 81 mask samples tested, respectively. We have shown that the masks tested had high VFE (>99% for surgical masks and respirators, ≥98% for material, and 87–97% for cloth masks) and that all experimental setups provided highly reproducible and reliable VFE results (coefficient of variation < 6%). Therefore, the VFE tests described in this study can be integrated into existing standards for mask testing.
Ključne besede: face masks, virus filtration efficiency, bacterial filtration efficiency, EN 14683:2019+AC:2019, air sampler
Objavljeno v DiRROS: 17.07.2024; Ogledov: 1; Prenosov: 0
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2. CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potatoTjaša Lukan, Florian Veillet, Maja Križnik, Anna Coll Rius, Tjaša Mahkovec Povalej, Karmen Pogačar, Katja Stare, Laura Chauvin, Jean-Eric Chauvin, Kristina Gruden, 2022, izvirni znanstveni članek Povzetek: MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.
Ključne besede: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Objavljeno v DiRROS: 17.07.2024; Ogledov: 3; Prenosov: 2
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3. Evaluation of methods and processes for robust monitoring of SARS-CoV-2 in wastewaterOlivera Maksimović, Živa Lengar, Zala Kogej Zwitter, Katarina Bačnik, Irena Bajde, Mojca Milavec, Anže Županič, Nataša Mehle, Denis Kutnjak, Maja Ravnikar, Ion Gutiérrez-Aguirre, 2022, izvirni znanstveni članek Povzetek: The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.
Ključne besede: waste water, method evolution, virus detection, SARS-CoV-2
Objavljeno v DiRROS: 17.07.2024; Ogledov: 0; Prenosov: 1
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4. Systematic comparison of nanopore and illumina sequencing for the detection of plant viruses and viroids using total RNA sequencing approachAnja Pecman, Ian Adams, Ion Gutiérrez-Aguirre, Adrian Fox, Neil Boonham, Maja Ravnikar, Denis Kutnjak, 2022, izvirni znanstveni članek Povzetek: High-throughput sequencing (HTS) has become an important tool for plant virus detection and discovery. Nanopore sequencing has been rapidly developing in the recent years and offers new possibilities for fast diagnostic applications of HTS. With this in mind, a study was completed, comparing the most established HTS platform (MiSeq benchtop sequencer—Illumina), with the MinION sequencer (Oxford Nanopore Technologies) for the detection of plant viruses and viroids. Method comparisons were performed on five selected samples, containing two viroids, which were sequenced using nanopore technology for the first time and 11 plant viruses with different genome organizations. For all samples, sequencing libraries for the MiSeq were prepared from ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) and for MinION sequencing, direct RNA sequencing of totRNA was used. Moreover, for one of the samples, which contained five different plant viruses and a viroid, three additional variations of sample preparation for MinION sequencing were also used: direct RNA sequencing of rRNA-depleted totRNA, cDNA-PCR sequencing of totRNA, and cDNA-PCR sequencing of rRNA-depleted totRNA. Whilst direct RNA sequencing of total RNA was the quickest of the tested approaches, it was also the least sensitive: using this approach, we failed to detect only one virus that was present in a sample at an extremely low titer. All other MinION sequencing approaches showed improved performance with outcomes similar to Illumina sequencing, with cDNA-PCR sequencing of rRNA-depleted totRNA showing the best performance amongst tested nanopore MinION sequencing approaches. Moreover, when enough sequencing data were generated, high-quality consensus viral genome sequences could be reconstructed from MinION sequencing data, with high identity to the ones generated from Illumina data. The results of this study implicate that, when an appropriate sample and library preparation are selected, nanopore MinION sequencing could be used for the detection of plant viruses and viroids with similar performance as Illumina sequencing. Taken as a balance of practicality and performance, this suggests that MinION sequencing may be an ideal tool for fast and affordable virus diagnostics.
Ključne besede: high-throughput sequencing, plant virus, viroid detection, comparison, nanopore MinION sequencing, illumina MiSeq sequencing
Objavljeno v DiRROS: 16.07.2024; Ogledov: 33; Prenosov: 9
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5. Development and validation of a one-step reverse transcription real-time PCR assay for simultaneous detection and identification of tomato mottle mosaic virus and tomato brown rugose fruit virusAntonio Tiberini, Ariana Manglli, Anna Taglienti, Ana Vučurović, Jakob Brodarič, Luca Ferretti, Marta Luigi, Andrea Gentili, Nataša Mehle, 2022, izvirni znanstveni članek Povzetek: Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.
Ključne besede: real-time PCR, tomato mottle mosaic virus, tomato brown rugose fruit virus, leaves detection, seeds detections, performance criteria
Objavljeno v DiRROS: 16.07.2024; Ogledov: 5; Prenosov: 5
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6. The physiological impact of GFLV virus infection on grapevine water status : first observationsAnastazija Jež Krebelj, Maja Cigoj, Marija Stele, Marko Chersicola, Maruša Pompe Novak, Paolo Sivilotti, 2022, izvirni znanstveni članek Povzetek: In a vineyard, grapevines are simultaneously exposed to combinations of several abiotic (drought, extreme temperatures, salinity) and biotic stresses (phytoplasmas, viruses, bacteria). With climate change, the incidences of drought in vine growing regions are increased and the host range of pathogens with increased chances of virulent strain development has expanded. Therefore, we studied the impact of the combination of abiotic (drought) and biotic (Grapevine fanleaf virus (GFLV) infection) stress on physiological and molecular responses on the grapevine of cv. Schioppettino by studying the influence of drought and GFLV infection on plant water status of grapevines, on grapevine xylem vessel occlusion, and on expression patterns of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1), 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), WRKY encoding transcription factor (WRKY54) and RD22-like protein (RD22) genes in grapevines. A complex response of grapevine to the combination of drought and GFLV infection was shown, including priming in the case of grapevine water status, net effect in the case of area of occluded vessels in xylem, and different types of interaction of both stresses in the case of expression of four abscisic acid-related genes. Our results showed that mild (but not severe) water stress can be better sustained by GFLV infection rather than by healthy vines. GFLV proved to improve the resilience of the plants to water stress, which is an important outcome to cope with the challenges of global warming.
Ključne besede: grapevine, water status, virus infection, GFLV, xylem vessel occlusion, gene expression
Objavljeno v DiRROS: 16.07.2024; Ogledov: 25; Prenosov: 8
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7. Hydrodynamic cavitation efficiently inactivates potato virus Y in waterArijana Filipić, Tadeja Lukežič, Katarina Bačnik, Maja Ravnikar, Meta Ješelnik, Tamara Košir, Martin Petkovšek, Mojca Zupanc, Matevž Dular, Ion Gutiérrez-Aguirre, 2022, izvirni znanstveni članek Povzetek: Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance of using proper strategies to treat recycled water in order to prevent the spread of the infectious agents. Emerging environmentally friendly methods such as hydrodynamic cavitation (HC) provide a great alternative for treating recycled water used for irrigation. In the experiments conducted in this study, laboratory HC based on Venturi constriction with a sample volume of 1 L was used to treat water samples spiked with purified PVY virions. The ability of the virus to infect plants was abolished after 500 HC passes, corresponding to 50 min of treatment under pressure difference of 7 bar. In some cases, shorter treatments of 125 or 250 passes were also sufficient for virus inactivation. The HC treatment disrupted the integrity of viral particles, which also led to a minor damage of viral RNA. Reactive species, including singlet oxygen, hydroxyl radicals, and hydrogen peroxide, were not primarily responsible for PVY inactivation during HC treatment, suggesting that mechanical effects are likely the driving force of virus inactivation. This pioneering study, the first to investigate eukaryotic virus inactivation by HC, will inspire additional research in this field enabling further improvement of HC as a water decontamination technology.
Ključne besede: hydrodynamic cavitation, potato virus Y, virus inactivation, water decontamination
Objavljeno v DiRROS: 16.07.2024; Ogledov: 35; Prenosov: 13
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8. Diversity and pathobiology of an ilarvirus unexpectedly detected in diverse plants and global sequencing dataMark Paul Selda Rivarez, Chantal Faure, Laurence Svanella-Dumas Svanella-Dumas, Anja Pecman, Magda Tušek-Žnidarič, Deborah Schönegger, Maja Ravnikar, Denis Kutnjak, 2023, izvirni znanstveni članek Ključne besede: phylogenetics, histopathology, pollen, virus diversity, virion morphology, symptomatology, virus transmission
Objavljeno v DiRROS: 12.07.2024; Ogledov: 45; Prenosov: 29
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9. Managing the deluge of newly discovered plant viruses and viroids : an optimized scientific and regulatory framework for their characterization and risk analysisNuria Fontdevila Pareta, Maryam Khalili, Ayoub Maachi, Mark Paul Selda Rivarez, Johan Rollin, Ferran Salavert Pamblanco, Coline Temple, Miguel A. Aranda, Denis Kutnjak, Maja Ravnikar, 2023, izvirni znanstveni članek Povzetek: The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.
Ključne besede: plant viruses and viroids, high throughput sequencing (HTS), biological characterization, plant health, regulatory agencies, Pest Risk Analysis (PRA), virus disease
Objavljeno v DiRROS: 12.07.2024; Ogledov: 45; Prenosov: 20
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10. Tomato brown rugose fruit virus in aqueous environments : survival and significance of water-mediated transmissionNataša Mehle, Katarina Bačnik, Irena Bajde, Jakob Brodarič, Adrian Fox, Ion Gutiérrez-Aguirre, Miha Kitek, Denis Kutnjak, Yue Lin Loh, Olivera Maksimović, Maja Ravnikar, Elise Vogel, Christine Vos, Ana Vučurović, 2023, izvirni znanstveni članek Povzetek: Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.
Ključne besede: tomato brown rugose fruit virus, tomato, hydroponics, water-linked epidemiology, survival
Objavljeno v DiRROS: 12.07.2024; Ogledov: 33; Prenosov: 19
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