Cold Plasma, a New Hope in the Field of Virus Inactivation

Pathogenic viruses are becoming an increasing burden for health, agriculture, and the global economy. Classic disinfection methods have several drawbacks, and innovative solutions for virus inactivation are urgently needed.CP can be used as an environmentally friendly tool for virus inactivation. It can inactivate different human, animal, and plant viruses in various matrices.When using CP for virus inactivation it is important to set the correct parameters and to choose treatment durations that allow particles to interact with the contaminated material.Reactive oxygen and/or nitrogen species have been shown to be responsible for virus inactivation through effects on capsid proteins and/or nucleic acids. The development of more accurate methods will provide information on which plasma particles are crucial in each experiment, and how exactly they affect viruses. Viruses can infect all cell-based organisms, from bacteria to humans, animals, and plants. They are responsible for numerous cases of hospitalization, many deaths, and widespread crop destruction, all of which result in an enormous medical, economical, and biological burden. Each of the currently used decontamination methods has important drawbacks. Cold plasma (CP) has entered this field as a novel, efficient, and clean solution for virus inactivation. We present recent developments in this promising field of CP-mediated virus inactivation, and describe the applications and mechanisms of the inactivation. This is particularly relevant because viral pandemics, such as COVID-19, highlight the need for alternative virus inactivation methods to replace, complement, or upgrade existing procedures. Viruses can infect all cell-based organisms, from bacteria to humans, animals, and plants. They are responsible for numerous cases of hospitalization, many deaths, and widespread crop destruction, all of which result in an enormous medical, economical, and biological burden. Each of the currently used decontamination methods has important drawbacks. Cold plasma (CP) has entered this field as a novel, efficient, and clean solution for virus inactivation. We present recent developments in this promising field of CP-mediated virus inactivation, and describe the applications and mechanisms of the inactivation. This is particularly relevant because viral pandemics, such as COVID-19, highlight the need for alternative virus inactivation methods to replace, complement, or upgrade existing procedures. Viruses are the most abundant and diverse microbes on our planet. They have inhabited the Earth for billions of years [1.Nasir A. Caetano-Anollés G. A phylogenomic data-driven exploration of viral origins and evolution.Sci. Adv. 2015; 1e1500527Crossref PubMed Scopus (131) Google Scholar], have adapted to various environments, and are now found across all ecosystems. Viruses have contributed to the evolution of life on Earth, and can be beneficial for preserving ecosystems and important natural Earth cycles such as the carbon cycle in the sea [2.Wilhelm S.W. Suttle C.A. Viruses and nutrient cycles in the sea aquatic food webs.Bioscience. 1999; 49: 781-788Crossref Scopus (845) Google Scholar]. On the other hand, pathogenic viruses cause tens to hundreds of millions of plant, animal, and human infections annually, which result in high crop losses and numerous deaths (Box 1). Therefore, inactivating harmful viruses is crucial for better quality of life.Box 1Viruses and Methods for Their DisinfectionViruses are microscopic agents that can infect all forms of cellular life. Their classification as living organisms has historically been a question of philosophical debate, but they are unquestionably one of the most powerful engines of evolution on the planet [51.Koonin E.V. Starokadomskyy P. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question.Stud. Hist. Phil. Biol. Biomed. Sci. 2016; 59: 125-134Crossref PubMed Scopus (76) Google Scholar]. Most viruses are not harmful, and some are even beneficial for their hosts [52.Roossinck M.J. Baz E.R. Symbiosis: viruses as intimate partners.Annu. Rev. Virol. 2017; 4: 123-139Crossref PubMed Scopus (63) Google Scholar]. In recent years viruses have been increasingly used to promote human wellbeing. For example, lentiviruses [53.Milone M.C. O'Doherty U. Clinical use of lentiviral vectors.Leukemia. 2018; 32: 1529-1541Crossref PubMed Scopus (437) Google Scholar] and adeno-associated viruses [54.Wang D. et al.Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378Crossref PubMed Scopus (946) Google Scholar] are being genetically engineered to formulate state-of-the-art gene therapies. Nevertheless, viruses have a bad reputation as causative agents of various human, animal, and plant diseases. This is no surprise because they have been the main players in numerous epidemics and pandemics throughout history (https://www.who.int/emergencies/diseases/managing-epidemics/en/). Several viral agents have contributed to the well-deserved 'biohazard' fame of viruses, including influenza, Ebola, HIV ,and coronavirus SARS-CoV-2. Despite not being such 'viral celebrities', waterborne viruses pose increasingly serious health and economic burdens in the present era that is threatened by climate change and the scarcity of potable water.Different physical and chemical treatments have traditionally been applied for inactivation of viruses. Chlorine, alcohols, acids, alkalis, and bleach are examples of chemical disinfectants, whereas UV irradiation, filtration, pressure, and temperature are physical treatments [55.Mehle N. et al.Water-mediated transmission of plant, animal, and human viruses.in: Malmstrom C.M. Advances in Virus Research. Academic Press, 2018: 85-128Google Scholar]. The method of choice depends on the matrix to be disinfected and on the virus targeted for inactivation. Waterborne viruses, including enteric viruses [56.Staggemeier R. et al.Animal and human enteric viruses in water and sediment samples from dairy farms.Agric. Water Manag. 2015; 152: 135-141Crossref Scopus (27) Google Scholar] and plant tobamoviruses [57.Zhang T. et al.RNA viral community in human feces: prevalence of plant pathogenic viruses.PLoS Biol. 2006; 4: 0108-0118Crossref Scopus (548) Google Scholar], are among the most stable of all viruses. To inactivate such stable viruses in a delicate matrix, the disinfection method needs to be strong enough to inactivate the virus but at the same time it needs to be nontoxic to maintain the quality and properties of the water. It is now known that chlorination, a traditionally used method for water disinfection, does not efficiently inactivate some viruses, and in the long term can pose a risk to human health through release of toxic byproducts [58.Lyon B.A. et al.Integrated chemical and toxicological investigation of UV-chlorine/ chloramine drinking water treatment.Environ. Sci. Technol. 2014; 48: 6743-6753Crossref PubMed Scopus (42) Google Scholar]. In recent years novel waterborne virus inactivation technologies have been developed, such as membrane filtration, reverse osmosis, UV and ozone treatments, and hydrodynamic cavitation, each of which has their own pros and cons. The frequent disadvantages of these technologies are cost inefficiency, scalability problems, and unsustainable power usage. Laboratory-scale studies suggest that CP has potential to overcome these problems, but confirmation will require studies focused on pilot or industrial scale deployment of plasma-based disinfection devices. Viruses are microscopic agents that can infect all forms of cellular life. Their classification as living organisms has historically been a question of philosophical debate, but they are unquestionably one of the most powerful engines of evolution on the planet [51.Koonin E.V. Starokadomskyy P. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question.Stud. Hist. Phil. Biol. Biomed. Sci. 2016; 59: 125-134Crossref PubMed Scopus (76) Google Scholar]. Most viruses are not harmful, and some are even beneficial for their hosts [52.Roossinck M.J. Baz E.R. Symbiosis: viruses as intimate partners.Annu. Rev. Virol. 2017; 4: 123-139Crossref PubMed Scopus (63) Google Scholar]. In recent years viruses have been increasingly used to promote human wellbeing. For example, lentiviruses [53.Milone M.C. O'Doherty U. Clinical use of lentiviral vectors.Leukemia. 2018; 32: 1529-1541Crossref PubMed Scopus (437) Google Scholar] and adeno-associated viruses [54.Wang D. et al.Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378Crossref PubMed Scopus (946) Google Scholar] are being genetically engineered to formulate state-of-the-art gene therapies. Nevertheless, viruses have a bad reputation as causative agents of various human, animal, and plant diseases. This is no surprise because they have been the main players in numerous epidemics and pandemics throughout history (https://www.who.int/emergencies/diseases/managing-epidemics/en/). Several viral agents have contributed to the well-deserved 'biohazard' fame of viruses, including influenza, Ebola, HIV ,and coronavirus SARS-CoV-2. Despite not being such 'viral celebrities', waterborne viruses pose increasingly serious health and economic burdens in the present era that is threatened by climate change and the scarcity of potable water. Different physical and chemical treatments have traditionally been applied for inactivation of viruses. Chlorine, alcohols, acids, alkalis, and bleach are examples of chemical disinfectants, whereas UV irradiation, filtration, pressure, and temperature are physical treatments [55.Mehle N. et al.Water-mediated transmission of plant, animal, and human viruses.in: Malmstrom C.M. Advances in Virus Research. Academic Press, 2018: 85-128Google Scholar]. The method of choice depends on the matrix to be disinfected and on the virus targeted for inactivation. Waterborne viruses, including enteric viruses [56.Staggemeier R. et al.Animal and human enteric viruses in water and sediment samples from dairy farms.Agric. Water Manag. 2015; 152: 135-141Crossref Scopus (27) Google Scholar] and plant tobamoviruses [57.Zhang T. et al.RNA viral community in human feces: prevalence of plant pathogenic viruses.PLoS Biol. 2006; 4: 0108-0118Crossref Scopus (548) Google Scholar], are among the most stable of all viruses. To inactivate such stable viruses in a delicate matrix, the disinfection method needs to be strong enough to inactivate the virus but at the same time it needs to be nontoxic to maintain the quality and properties of the water. It is now known that chlorination, a traditionally used method for water disinfection, does not efficiently inactivate some viruses, and in the long term can pose a risk to human health through release of toxic byproducts [58.Lyon B.A. et al.Integrated chemical and toxicological investigation of UV-chlorine/ chloramine drinking water treatment.Environ. Sci. Technol. 2014; 48: 6743-6753Crossref PubMed Scopus (42) Google Scholar]. In recent years novel waterborne virus inactivation technologies have been developed, such as membrane filtration, reverse osmosis, UV and ozone treatments, and hydrodynamic cavitation, each of which has their own pros and cons. The frequent disadvantages of these technologies are cost inefficiency, scalability problems, and unsustainable power usage. Laboratory-scale studies suggest that CP has potential to overcome these problems, but confirmation will require studies focused on pilot or industrial scale deployment of plasma-based disinfection devices. Viruses can be transmitted directly from one infected individual to another or indirectly via contaminated intermediates such as surfaces, objects, air, food, and water. Transmission via contaminated surfaces and aerosols has shown to be of great importance in the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [3.van Doremalen N. et al.Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1.N. Engl. J. Med. 2020; 382: 1564-1567Crossref PubMed Scopus (6351) Google Scholar]. Water is also an increasingly important transmission route for pathogenic viruses. This has arisen from global climate change and the continued increase in water demand, combined with inefficient virus removal by traditional water treatments and reuse of wastewater for irrigation purposes [4.Shrestha S. et al.Virological quality of irrigation water sources and pepper mild mottle virus and tobacco mosaic virus as index of pathogenic virus contamination level.Food Environ. Virol. 2018; 10: 107-120Crossref PubMed Scopus (47) Google Scholar,5.Mehle N. Ravnikar M. Plant viruses in aqueous environment – survival, water mediated transmission and detection.Water Res. 2012; 46: 4902-4917Crossref PubMed Scopus (61) Google Scholar]. Pathogenic waterborne viruses are important contributors to one of the most important global risks we are facing today, the scarcity of potable water [6.Franco E.G. et al.The Global Risks Report 2020.15th edn. World Economic Forum, 2020Google Scholar]. Various virus inactivation (see Glossary) methods are used to prevent viral spread in different matrices, but unfortunately the ideal method has yet to be discovered (Box 1). Thus, there is an urgent need for an environmentally friendly treatment that produces neither waste nor toxic byproducts, does not use toxic chemicals, is easy and safe to work with, and is also efficient in terms of virus inactivation. The emergence of CP treatments for virus inactivation aims to provide a solution to all of these features. Plasma is the fourth state of matter. It is a partially or fully ionized gas where the atoms and/or molecules are stripped of their outer-shell electrons (Box 2) [7.Filipić A. et al.Cold atmospheric plasma as a novel method for inactivation of potato virus Y in water samples.Food Environ. Virol. 2019; 11: 220-228Crossref PubMed Scopus (29) Google Scholar]. Among its complex constituents, the emission of UV radiation and reactive oxygen and/or nitrogen species (RONS) have the most important antimicrobial properties [8.Guo J. et al.Bactericidal effect of various non-thermal plasma agents and the influence of experimental conditions in microbial inactivation: a review.Food Control. 2015; 50: 482-490Crossref Scopus (151) Google Scholar]. UV can damage nucleic acids [9.US Environmental Protection Agency Office of Water Ultraviolet Disinfection Guidance Manual for the Final Long Term 2 Enhanced Surface Water Treatment Rule. US EPA, 2006Google Scholar], whereas RONS can oxidize nucleic acids, proteins, and lipids, with different affinities that depend on the species [10.Mittler R. ROS are good.Trends Plant Sci. 2017; 22: 11-19Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar]. These inherent properties of plasma, and more specifically of CP, have motivated extensive studies on the use of CP for inactivation of various pathogenic microorganisms. The main target has been bacteria, with investigations across different fields such as food production [11.Bourke P. et al.The potential of cold plasma for safe and sustainable food production.Trends Biotechnol. 2018; 36: 615-626Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar], medicine, and dentistry [12.Sakudo A. et al.Disinfection and sterilization using plasma technology: fundamentals and future perspectives for biological applications.Int. J. Mol. Sci. 2019; 20: 5216Crossref PubMed Scopus (143) Google Scholar]. These have even extended to oncotherapy applications, where cancer cells are targeted instead of pathogenic microorganisms [13.Dai X. et al.The emerging role of gas plasma in oncotherapy.Trends Biotechnol. 2018; 36: 1183-1198Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar].Box 2Let's Talk about PlasmaPlasma is the most abundant state of matter and comprises 99% of the visible universe. The sun and other stars, nebulae, solar winds, lightning, and aurora borealis are all in the plasma state. Plasma TVs as well as neon and fluorescent lights are the best-known man-made uses of plasma. Generally, plasma contains free electrons, atoms, and molecules in neutral, ionized, and/or excited states (including ROS/RNS). Plasma of many gases represents an extensive source of UV and vacuum UV radiation [59.Machala Z. Pavlovich M.J. A new phase in applied biology.Trends Biotechnol. 2018; 36: 577-578Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. The possibility to use a particular or a combination of constituents makes plasma a unique material-treatment technique.Plasma can be roughly divided into thermal or equilibrium plasma, where all particles have approximately the same temperature (average kinetic energy of random motion), and nonthermal, nonequilibrium, or CP, where light electrons have much higher temperatures compared with heavy atoms and molecules, which often remain close to room temperature. In other words, CP is at the point of application at room temperature, and is therefore suitable for treating diverse biological material including solids, liquids, and aerosols. CP can be further classified into low pressure and atmospheric pressure. The latter is limited to the volume where there are large electric fields, whereas low-pressure plasma spreads in a large volume [60.Mozetič M. et al.Introduction to plasma and plasma diagnostics.in: Thomas S. Non-Thermal Plasma Technology for Polymeric Materials: Applications In Composites, Nanostructured Materials and Biomedical Fields. Elsevier, 2019: 23-65Crossref Scopus (12) Google Scholar]. CP is usually sustained with an electrical discharge. The gas temperature is usually almost unaffected, but the chemical reactivity is vast compared with the source gas because of the presence of reactive species. In most cases of virus inactivation, atmospheric pressure plasma has been used because of practical considerations ([61.Ehlbeck J. et al.Low temperature atmospheric pressure plasma sources for microbial decontamination.J. Phys. D. Appl. Phys. 2011; 44013002Crossref Scopus (581) Google Scholar] for more information on various plasma sources used in microbial decontamination).Plasmas are used in various industries, mainly for tailoring the surfaces of solids (e.g., oxidation, cleaning, nanostructuring, binding different atom/molecule groups), but also for the destruction of microorganisms such as viruses. Plasma can also be used for treatment of liquids; however, inactivation of viruses in liquid media is more challenging than for surfaces because plasma cannot be sustained in liquids, and is only present in gaseous bubbles inside the liquid or above the liquid surface. Depending on the place of their generation, RONS interact with either the bubble surface or the surface of the liquid, where many dissolve. They can then diffuse within the liquid, and may eventually interact with the virus. Furthermore, UV radiation penetrates liquids with a penetration depth that depends greatly on the wavelength, and the concentration, and type of impurities [62.Bruggeman P.J. et al.Plasma-liquid interactions: a review and roadmap.Plasma Sources Sci. Technol. 2016; 25053002Crossref Scopus (1024) Google Scholar]. There are various techniques for measuring both long- and short-lived RONS in liquids [63.Labay C. et al.Production of reactive species in alginate hydrogels for cold atmospheric plasma-based therapies.Sci. Rep. 2019; 9: 1-12Crossref PubMed Scopus (34) Google Scholar], but these are not used frequently by researchers working on the destruction of viruses. Many authors state the discharge parameters (voltage, current, power) rather than the plasma parameters (concentration of reactive species) which are necessary to compare various plasma sources. The plasma–virus scientific niche is therefore in its infancy at present. Plasma is the most abundant state of matter and comprises 99% of the visible universe. The sun and other stars, nebulae, solar winds, lightning, and aurora borealis are all in the plasma state. Plasma TVs as well as neon and fluorescent lights are the best-known man-made uses of plasma. Generally, plasma contains free electrons, atoms, and molecules in neutral, ionized, and/or excited states (including ROS/RNS). Plasma of many gases represents an extensive source of UV and vacuum UV radiation [59.Machala Z. Pavlovich M.J. A new phase in applied biology.Trends Biotechnol. 2018; 36: 577-578Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. The possibility to use a particular or a combination of constituents makes plasma a unique material-treatment technique. Plasma can be roughly divided into thermal or equilibrium plasma, where all particles have approximately the same temperature (average kinetic energy of random motion), and nonthermal, nonequilibrium, or CP, where light electrons have much higher temperatures compared with heavy atoms and molecules, which often remain close to room temperature. In other words, CP is at the point of application at room temperature, and is therefore suitable for treating diverse biological material including solids, liquids, and aerosols. CP can be further classified into low pressure and atmospheric pressure. The latter is limited to the volume where there are large electric fields, whereas low-pressure plasma spreads in a large volume [60.Mozetič M. et al.Introduction to plasma and plasma diagnostics.in: Thomas S. Non-Thermal Plasma Technology for Polymeric Materials: Applications In Composites, Nanostructured Materials and Biomedical Fields. Elsevier, 2019: 23-65Crossref Scopus (12) Google Scholar]. CP is usually sustained with an electrical discharge. The gas temperature is usually almost unaffected, but the chemical reactivity is vast compared with the source gas because of the presence of reactive species. In most cases of virus inactivation, atmospheric pressure plasma has been used because of practical considerations ([61.Ehlbeck J. et al.Low temperature atmospheric pressure plasma sources for microbial decontamination.J. Phys. D. Appl. Phys. 2011; 44013002Crossref Scopus (581) Google Scholar] for more information on various plasma sources used in microbial decontamination). Plasmas are used in various industries, mainly for tailoring the surfaces of solids (e.g., oxidation, cleaning, nanostructuring, binding different atom/molecule groups), but also for the destruction of microorganisms such as viruses. Plasma can also be used for treatment of liquids; however, inactivation of viruses in liquid media is more challenging than for surfaces because plasma cannot be sustained in liquids, and is only present in gaseous bubbles inside the liquid or above the liquid surface. Depending on the place of their generation, RONS interact with either the bubble surface or the surface of the liquid, where many dissolve. They can then diffuse within the liquid, and may eventually interact with the virus. Furthermore, UV radiation penetrates liquids with a penetration depth that depends greatly on the wavelength, and the concentration, and type of impurities [62.Bruggeman P.J. et al.Plasma-liquid interactions: a review and roadmap.Plasma Sources Sci. Technol. 2016; 25053002Crossref Scopus (1024) Google Scholar]. There are various techniques for measuring both long- and short-lived RONS in liquids [63.Labay C. et al.Production of reactive species in alginate hydrogels for cold atmospheric plasma-based therapies.Sci. Rep. 2019; 9: 1-12Crossref PubMed Scopus (34) Google Scholar], but these are not used frequently by researchers working on the destruction of viruses. Many authors state the discharge parameters (voltage, current, power) rather than the plasma parameters (concentration of reactive species) which are necessary to compare various plasma sources. The plasma–virus scientific niche is therefore in its infancy at present. Plasma-mediated virus inactivation is a relatively young field of research (reviewed in [14.Puligundla P. Mok C. Non-thermal plasmas (NTPs) for inactivation of viruses in abiotic environment.Res. J. Biotechnol. 2016; 11: 91-96PubMed Google Scholar,15.Weiss M. et al.Virucide properties of cold atmospheric plasma for future clinical applications.J. Med. Virol. 2017; 89: 952-959Crossref PubMed Scopus (39) Google Scholar]) which started only about 20 years ago [16.Kelly-Wintenberg K. et al.Use of a one atmosphere uniform glow discharge plasma to kill a broad spectrum of microorganisms.J. Vac. Sci. Technol. A. 1999; 17: 1539-1544Crossref Scopus (179) Google Scholar]. This is despite the decades-old knowledge that ozone, that is usually synthesized from O2 subjected to plasma conditions, can inactivate viruses [17.Burleson G.R. et al.Inactivation of viruses and bacteria by ozone, with and without sonication.Appl. Microbiol. 1975; 29: 340-344Crossref PubMed Google Scholar]. However, over the past few years the number of publications in the CP–virus field has doubled, and research has expanded from only defining the virucidal properties of plasma to describing its modes of inactivation. This review offers a comprehensive overview of the latest progress and achievements in the CP–virus field. We also describe and discuss the modes of CP-mediated virus inactivation and the reactive species that are responsible. Almost every study on CP inactivation of viruses is unique because they either use a specific plasma source [e.g., dielectric barrier discharge (DBD), plasma (micro)jets] (Figure 1) with different characteristics (e.g., power, gas, treatment time) or they deal with the treatment of different liquid volumes (from microliters to several milliliters), matrices (e.g., water, other solutions, surfaces, cells), and viruses (surrogates of human viruses, human, animal, and plant viruses). Such wide diversity makes it difficult to directly compare these studies and to define the mechanistic conclusions or any universal inactivation parameters. To simplify these complexities, we consider here the individual types of viruses that have been subjected to CP treatments. A complete list of the treatments published to date is given in Table S1, Table S2 in the supplemental information online. CP treatments have been often focused on enteric viruses such as norovirus, adenovirus, and hepatitis A virus. These are the leading causes of acute gastroenteritis, the second most common infectious disease worldwide, which is responsible for high levels of hospitalization and mortality [18.McMinn B.R. et al.Bacteriophages as indicators of faecal pollution and enteric virus removal.Lett. Appl. Microbiol. 2017; 65: 11-26Crossref PubMed Scopus (42) Google Scholar]. Working with human viruses can pose serious health hazards, and such studies require specialized laboratories and equipment. Moreover, infectivity assessments of important enteric viruses, such as norovirus, have been limited owing to a lack of cultivation methods [19.Cromeans T. et al.Comprehensive comparison of cultivable norovirus surrogates in response to different inactivation and disinfection treatments.Appl. Environ. Microbiol. 2014; 80: 5743-5751Crossref PubMed Scopus (151) Google Scholar]. For these reasons, these viruses are often replaced by surrogate viruses. Animal viruses such as feline calicivirus (FCV), murine norovirus (MNV), and Tulane virus (TV) have been used as surrogates for norovirus owing to their similar sizes, morphologies, and genetic material. Furthermore, these surrogate viruses are easy to culture/reproduce, and are safe to work with [19.Cromeans T. et al.Comprehensive comparison of cultivable norovirus surrogates in response to different inactivation and disinfection treatments.Appl. Environ. Microbiol. 2014; 80: 5743-5751Crossref PubMed Scopus (151) Google Scholar]. Opinions are divided over which of these surrogate viruses best resembles the stability of norovirus, and the final choice strongly depends on the inactivation method used and the environmental properties [13.Dai X. et al.The emerging role of gas plasma in oncotherapy.Trends Biotechnol. 2018; 36: 1183-1198Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14.Puligundla P. Mok C. Non-thermal plasmas (NTPs) for inactivation of viruses in abiotic environment.Res. J. Biotechnol. 2016; 11: 91-96PubMed Google Scholar, 15.Weiss M. et al.Virucide properties of cold atmospheric plasma for future clinical applications.J. Med. Virol. 2017; 89: 952-959Crossref PubMed Scopus (39) Google Scholar]. In addition to animal viruses, bacteriophages (viruses that infect bacteria) can be used as surrogates for enteric viruses and other human pathogens (Box 3).Box 3Bacteriophages as Surrogates, and an Alternative CP TreatmentBacteriophages are the first choice in many studies to establish proof of concept for virus inactivation methods because of their many advantages. They are relatively inexpensive to culture/produce, easy and safe to work with, they can be produced in large quantities, and plaque-based infectivity assays are time-efficient [64.McMinn B.R. et al.Bacteriophages as indicators of faecal pollution and enteric virus removal.Lett. Appl. Microbiol. 2017; 65: 11-26Crossref PubMed Scopus (112) Google Scholar]. However, care must be taken when interpreting the results because they do not always correlate with the response of the actual virus to the inactivation method.The first study that triggered the expansion of the plasma–virus field was conducted on bacteriophages [16.Kelly-Wintenberg K. et al.Use of a one atmosphere uniform glow discharge plasma to kill a broad spectrum of microorganisms.J. Vac. Sci. Technol. A. 1999; 17: 1539-1544Crossref Scopus (179) Google Scholar]. In recent years, bacteriophages