This scientific paper contains an overview of the latest research results in the field of virus inactivation with cold gaseous plasma, which is a new, environmentally friendly method for virus inactivation. The reader is introduced to the successful inactivation of many human, animal, and plant viruses in or on various matrices, from liquids and air to model surfaces and foods. In addition, the mechanisms of inactivation are described, which includes descriptions of the major plasma particles involved in virus inactivation and their effects on different parts of the virus. All this makes this publication the first comprehensive review in the field of virus inactivation with cold plasma. For months after its publication, the article was counted among the most read articles in the renowned journal Trends and Biotechnology, IF= 14.3. The article also includes our research in cold plasma virus inactivation, which we have presented at numerous national and international conferences, including invited talks. We have received several awards for our presentations, which confirms the importance of the topic and the quality of the results.
COBISS.SI-ID: 33308199
We have studied biology, evolution and epidemiology of plant viruses and establish state-of-the art methods for their detection and presented our work as invited lectures on the biggest conference on the topic of plant pathology (Kutnjak, Denis, Pecman, Anja, Bačnik, Katarina, Gutiérrez-Aguirre, Ion, Mehle, Nataša, Tušek-Žnidarič, Magda, Ravnikar, Maja, IPPC, COBISS ID: 4793167) and plant virus epidemiology (Ravnikar, Maja, Kutnjak, Denis, Bačnik, Katarina, Pecman, Anja, Mehle, Nataša, Kogovšek, Polona, Tušek-Žnidarič, Magda, Gutiérrez-Aguirre, Ion, IPVE, COBISS ID: 5322575). In a highly cited paper (Pecman, Anja, Kutnjak, Denis, Gutiérrez-Aguirre, Ion, Adams, Ian, Fox, Adrian, Boonham, Neil, Ravnikar, Maja, Frontiers in Microbiology, COBISS ID: 4476495, IF=4.019), we have for the first time systematically compared different approaches for plant virus detection using high-throughput sequencing. We have also used this method to study experimental evolution of a virus (Kutnjak, Denis, Elena, Santiago F., Ravnikar, Maja, Journal of Virology, COBISS ID: 4390479, IF=4.368), and, in a recent study, to investigate an understudied part of wastewater virome – plant viruses. To determine the abundance, diversity and biological relevance of plant viruses in wastewater, we applied an optimized virus concentration method followed by high-throughput sequencing and infectivity assays. We detected representatives of 47 plant virus species, including emerging crop threats. We also demonstrated infectivity for pathogenic and economically relevant plant viruses from the genus Tobamovirus (family Virgaviridae), which remain infective even after conventional wastewater treatment. These results demonstrate the potential of metagenomics to capture the diversity of plant viruses circulating in the environment and expose the potential risk of the uncontrolled use of reclaimed water for irrigation. This study was selected among best scientific achievements (two of the best in the field of Biotechnology) by Slovenian Research Agency in 2020 (Excellence in Science program).
COBISS.SI-ID: 5318735
Waterborne viruses pose a high health, environmental, and economic risk, making their inactivation critical. This study addressed this issue and described for the first time the inactivation of a waterborne virus, pepper mild mottle virus (PMMoV), with cold atmospheric plasma (CAP). This work was also one of the first studies on inactivation of plant viruses using CAP and inactivation of viruses in larger volumes of water. In this study, we investigated the effects of CAP on the highly resistant tobamovirus, PMMoV. PMMoV is not only important as an agricultural pest where it causes high losses in pepper crops, but it also represents an important link between problematic tobamoviruses and enteric viruses. We achieved inactivation of this stable virus after only a few minutes of CAP treatment. In this study, we also investigated the effects of CAP treatment on viral proteins and RNA, as well as the possible genotoxicity and cytotoxicity of CAP -treated water. We demonstrated that CAP can affect both viral proteins and RNA without releasing toxic by-products into the water medium. Therefore, CAP represents an alternative method for water decontamination that can inactivate even the most resistant waterborne viruses.
COBISS.SI-ID: 49725955
One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. During optimization of the processes, analytical methods used for evaluation of the individual process step for characterization of the virus, play an important role. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
COBISS.SI-ID: 5125199
This publication represents pioneering work on the inactivation of PVY with cold atmospheric plasma (CAP), and was one of the first to deal with the inactivation of plant viruses with CAP, and with the inactivation of viruses in larger quantities of water. In this study, the particularly aggressive PVY strain was used: PVYNTN. It can infect different plants from the family Solanaceae, including pepper, tomato, eggplant, tobacco and potato. It is the most economically important viral potato pathogen, and it can cause 85% production losses. This is a major problem, as potato is one of the most important crops in the world. As PVY can cause high crop losses, it is important to prevent its transmission by water. We used two types of virus-infected water samples i.e., nutrient solution with high (i.e., more polluted samples) and with low (i.e., less polluted samples) organic background. With this, we wanted to observe the effect of different organic backgrounds on virus inactivation. Additionally, examined the mechanisms of inactivation. These included a description of the CAP properties that were linked to PVY inactivation, and their effects on the PVY RNA. We showed that reactive oxygen species have important roles in the virus inactivation, whereas UV radiation does not. We also observed that the CAP treatment can degraded the viral RNA. These findings served as an important starting point for new experiments in the field of water decontamination with CAP.
COBISS.SI-ID: 5060943