1. 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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2. 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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3. 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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4. Benthic sediment as stores and sources of bacteria and viruses in streams : a comparison of baseflow vs. stormflow longitudinal transport and residence timesJennifer D Drummond, José Gonçalves, Tomás Aquino, Susan A. Bernal, Esperança Gacia, Ion Gutiérrez-Aguirre, Valentina Turk, Maja Ravnikar, Stefan Krause, Eugenia Martí Roca, 2023, izvirni znanstveni članek Povzetek: The presence of bacteria and viruses in freshwater represents a global health risk. The substantial spatial and temporal variability of microbes leads to difficulties in quantifying the risks associated with their presence in freshwater. Fine particles, including bacteria and viruses are transported and accumulated into shallow streambed (i.e., benthic) sediment, delaying the downstream transmission during baseflow conditions but contributing to their resuspension and transport downstream during stormflow events. Direct measurements of pathogen accumulation in benthic sediments are rare. Until now, the dynamic role of benthic sediment as both a store and source of microbes, has not been quantified. In this study, we analyze microbial abundance in benthic sediment along a 1 km reach of an intermittent Mediterranean stream receiving inputs from the effluent of a wastewater treatment plant, a known point source of microbes in streams. We sampled benthic sediment during a summer drought when the wastewater effluent constituted 100 % of the stream flow, and thus, large accumulation and persistence of pathogens along the streambed was expected. We measured the abundance of total bacteria, Escherichia coli (as a fecal indicator), and presence of enteric rotavirus (RoV) and norovirus (NoV). The abundance of E. coli, based on qPCR detection, was high (4.99∙102 gc /cm2) along the first 100 m downstream of the wastewater effluent input and in general decreased with distance from the source, with presence of RoV and NoV along the study reach. A particle tracking model was applied, that uses stream water velocity as an input, and accounts for microbial exchange into, immobilization, degradation, and resuspension out of benthic sediment during baseflow and stormflow. Rates of exchange into benthic sediment were 3 orders of magnitude higher during stormflow, but residence times were proportionately lower, resulting in increased longitudinal connectivity from up to downstream during stormflow. Model simulations demonstrated mechanistically how the rates of exchange into and out of the benthic sediment resulted in benthic sediment to act as a store during baseflow and a source during stormflow.
Ključne besede: E. coli, norovirus, rotavirus, benthic sediment, pathogen transport, microbial risk
Objavljeno v DiRROS: 12.07.2024; Ogledov: 55; Prenosov: 20
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5. 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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6. Virome analysis of irrigation water sources provides extensive insights into the diversity and distribution of plant viruses in agroecosystemsOlivera Maksimović, Katarina Bačnik, Mark Paul Selda Rivarez, Ana Vučurović, Nataša Mehle, Maja Ravnikar, Ion Gutiérrez-Aguirre, Denis Kutnjak, 2024, izvirni znanstveni članek Povzetek: Plant viruses pose a significant threat to agriculture. Several are stable outside their hosts, can enter water bodies and remain infective for prolonged periods of time. Even though the quality of irrigation water is of increasing importance in the context of plant health, the presence of plant viruses in irrigation waters is understudied. In this study, we conducted a large-scale high-throughput sequencing (HTS)-based virome analysis of irrigation and surface water sources to obtain complete information about the abundance and diversity of plant viruses in such waters. We detected nucleic acids of plant viruses from 20 families, discovered several novel plant viruses from economically important taxa, like Tobamovirus and observed the influence of the water source on the present virome. By comparing viromes of water and surrounding plants, we observed presence of plant viruses in both compartments, especially in cases of large-scale outbreaks, such as that of tomato mosaic virus. Moreover, we demonstrated that water virome data can extensively inform us about the distribution and diversity of plant viruses for which only limited information is available from plants. Overall, the results of the study provided extensive insights into the virome of irrigation waters from the perspective of plant health. It also suggested that an HTS-based water virome surveillance system could be used to detect potential plant disease outbreaks and to survey the distribution and diversity of plant viruses in the ecosystem.
Ključne besede: plant viruses, environmental water testing, high-throughput sequencing, agroecosystems, irrigation water, virome
Objavljeno v DiRROS: 29.03.2024; Ogledov: 312; Prenosov: 139
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7. In-depth study of tomato and weed viromes reveals undiscovered plant virus diversity in an agroecosystemMark Paul Selda Rivarez, Anja Pecman, Katarina Bačnik, Olivera Maksimović, Ana Vučurović, Gabrijel Seljak, Nataša Mehle, Ion Gutiérrez-Aguirre, Maja Ravnikar, Denis Kutnjak, 2023, izvirni znanstveni članek Povzetek: Background: In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles of viruses in agroecosystems. Such unbiased exploration is available through viromics, which could generate biological and ecological insights from immense high-throughput sequencing (HTS) data. Results: Here, we implemented HTS-based viromics to explore viral diversity in tomatoes and weeds in farming areas at a nation-wide scale. We detected 125 viruses, including 79 novel species, wherein 65 were found exclusively in weeds. This spanned 21 higher-level plant virus taxa dominated by Potyviridae, Rhabdoviridae, and Tombusviridae, and four non-plant virus families. We detected viruses of non-plant hosts and viroid-like sequences and demonstrated infectivity of a novel tobamovirus in plants of Solanaceae family. Diversities of predominant tomato viruses were variable, in some cases, comparable to that of global isolates of the same species. We phylogenetically classified novel viruses and showed links between a subgroup of phylogenetically related rhabdoviruses to their taxonomically related host plants. Ten classified viruses detected in tomatoes were also detected in weeds, which might indicate possible role of weeds as their reservoirs and that these viruses could be exchanged between the two compartments. Conclusions: We showed that even in relatively well studied agroecosystems, such as tomato farms, a large part of very diverse plant viromes can still be unknown and is mostly present in understudied non-crop plants. The overlapping presence of viruses in tomatoes and weeds implicate possible presence of virus reservoir and possible exchange between the weed and crop compartments, which may influence weed management decisions. The observed variability and widespread presence of predominant tomato viruses and the infectivity of a novel tobamovirus in solanaceous plants, provided foundation for further investigation of virus disease dynamics and their effect on tomato health. The extensive insights we generated from such in-depth agroecosystem virome exploration will be valuable in anticipating possible emergences of plant virus diseases and would serve as baseline for further post-discovery characterization studies.
Ključne besede: tomato, weed, virus, viroid, virome, virus discovery, virus diversity, phylogenetics, metagenomics, viromics
Objavljeno v DiRROS: 13.04.2023; Ogledov: 741; Prenosov: 175
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