Mechanism Of Virus Inactivation Research Articles

Quantitative risk assessment model of the presence of porcine epidemic diarrhea and African swine fever viruses in spray-dried porcine plasma.

There are no microbiological regulatory limits for viruses in animal feed and feed ingredients. A performance objective (PO) was proposed in this study to manufacture a spray-dried porcine plasma (SDPP) batch absent of any infectious viral particles. The PO levels of -7.0, -7.2, and -7.3 log TCID50/g in SDPP were estimated for three batch sizes (10, 15, and 20 tons). A baseline survey on the presence of porcine epidemic diarrhea virus (PEDV) in raw porcine plasma revealed a concentration of -1.0 ± 0.6 log TCID50/mL as calculated using a TCID50-qPCR derived standard curve. The mean African swine fever virus (ASFV) concentration in raw plasma was estimated to be 0.6 log HAD50/mL (0.1-1.4, 95% CI) during a pre-clinical scenario (collected from asymptomatic and undetected viremic pigs). Different processing scenarios (baseline: spray-drying + extended storage) and baseline + ultraviolet (UV) radiation were evaluated to meet the PO levels proposed in this study. The baseline and baseline + UV processing scenarios were >95 and 100% effective in achieving the PO for PEDV by using different batch sizes. For the ASFV in SDPP during a pre-clinical scenario, the PO compliance was 100% for all processing scenarios evaluated. Further research is needed to determine the underlying mechanisms of virus inactivation in feed storage to further advance the implementation of feed safety risk management efforts globally.

The role of ion dissolution in metal and metal oxide surface inactivation of SARS-CoV-2.

Anti-viral surface coatings are under development to prevent viral fomite transmission from high-traffic touch surfaces in public spaces. Copper's anti-viral properties have been widely documented, but the anti-viral mechanism of copper surfaces is not fully understood. We screened a series of metal and metal oxide surfaces for anti-viral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19). Copper and copper oxide surfaces exhibited superior anti-SARS-CoV-2 activity; however, the level of anti-viral activity was dependent on the composition of the carrier solution used to deliver virus inoculum. We demonstrate that copper ions released into solution from test surfaces can mediate virus inactivation, indicating a copper ion dissolution-dependent anti-viral mechanism. The level of anti-viral activity is, however, not dependent on the amount of copper ions released into solution per se. Instead, our findings suggest that degree of virus inactivation is dependent on copper ion complexation with other biomolecules (e.g., proteins/metabolites) in the virus carrier solution that compete with viral components. Although using tissue culture-derived virus inoculum is experimentally convenient to evaluate the anti-viral activity of copper-derived test surfaces, we propose that the high organic content of tissue culture medium reduces the availability of "uncomplexed" copper ions to interact with the virus, negatively affecting virus inactivation and hence surface anti-viral performance. We propose that laboratory anti-viral surface testing should include virus delivered in a physiologically relevant carrier solution (saliva or nasal secretions when testing respiratory viruses) to accurately predict real-life surface anti-viral performance when deployed in public spaces.IMPORTANCEThe purpose of evaluating the anti-viral activity of test surfaces in the laboratory is to identify surfaces that will perform efficiently in preventing fomite transmission when deployed on high-traffic touch surfaces in public spaces. The conventional method in laboratory testing is to use tissue culture-derived virus inoculum; however, this study demonstrates that anti-viral performance of test copper-containing surfaces is dependent on the composition of the carrier solution in which the virus inoculum is delivered to test surfaces. Therefore, we recommend that laboratory surface testing should include virus delivered in a physiologically relevant carrier solution to accurately predict real-life test surface performance in public spaces. Understanding the mechanism of virus inactivation is key to future rational design of improved anti-viral surfaces. Here, we demonstrate that release of copper ions from copper surfaces into small liquid droplets containing SARS-CoV-2 is a mechanism by which the virus that causes COVID-19 can be inactivated.

Investigating the Effects of Cold Plasma on Cancer Cell Migration in the Presence of a Static Magnetic Field

Cold plasma is used in a wide range of applications [1]–[3] One of the primary uses of plasma is in the field of medicine. Moreover, many techniques have been suggested to enhance plasma performance, among which the inclusion of an external static magnetic field is one [4], [5]. This paper used a low-power (20-watt) RF 13.56 MHz plasma to investigate the effect of cold plasma on cancer cells' migration rate in the presence of a static magnetic field. The result of the scratch healing assay is shown in Figure 1 (Ⅰ and Ⅱ). The results are evaluated after 24 hours. Figure 1 (Ⅰ-A) is before plasma treatment (control), (Ⅰ-B) is without treatment, (Ⅰ-C) is 15 seconds of plasma treatment, and (Ⅰ-D) is plasma and SMF treatment. The area of free space in the created gap has been evaluated by image processing software. The percentage of free space compared to the total space is specified in the diagram (Figure1-Ⅱ). As shown in the figure, the empty space for the state where the plasma is combined with the magnetic field is twice the state of the plasma alone. One of the main components of plasma that changes the presence of a magnetic field is the density and temperature of electrons in cold plasma. The electron temperature was evaluated based on the Boltzmann plot method, and the electron density measurement was calculated using the Saha-Boltzmann equation (figure 1-Ⅲ). The Boltzmann diagram is drawn using the used plasma spectrum, which was used in the previous work [6]. The calculation indicates that the electron temperature Te and electron density ne is estimated as 1.04 eV and for the absence of SMF, and 1.24 eV and for the presence of SMF, respectively Although several factors are involved in the effect of plasma, in this work, the temperature and electron density of the plasma were evaluated. Because apart from the direct impact on the target, the electron also affects the density of the ions. The findings indicate that the presence of a stationary magnetic field in close proximity to a plasma significantly regulated the migratory rate of cancer cells.Figure 1. (Ⅰ) scratch healing assay, (Ⅱ) percentage of gap for treatments, (Ⅲ) electron temperature calculation with Boltzmann plot.[1] S. Padureanu et al., “Non-Thermal Plasma-Activated Water: A Cytogenotoxic Potential on Triticum aestivum,” Agronomy, vol. 13, no. 2, 2023, doi: 10.3390/agronomy13020459.[2] V. Ioannou and S. Laizet, “Numerical investigation of plasma-controlled turbulent jets for mixing enhancement,” Int. J. Heat Fluid Flow, vol. 70, no. October 2017, pp. 193–205, 2018, doi: 10.1016/j.ijheatfluidflow.2018.02.008.[3] L. Guo et al., “Mechanism of virus inactivation by cold atmospheric-pressure plasma and plasmaactivated water,” Appl. Environ. Microbiol., vol. 84, no. 17, 2018, doi: 10.1128/AEM.00726-18.[4] X. Cheng et al., “Enhancing cold atmospheric plasma treatment of cancer cells by static magnetic field,” Bioelectromagnetics, vol. 38, no. 1, pp. 53–62, 2017, doi: 10.1002/bem.22014.[5] R. Mehrabifard, H. Mehdian, K. Hajisharifi, and E. Amini, “Improving Cold Atmospheric Pressure Plasma Efficacy on Breast Cancer Cells Control-Ability and Mortality Using Vitamin C and Static Magnetic Field,” Plasma Chem. Plasma Process., vol. 40, no. 2, pp. 511–526, Nov. 2020, doi: 10.1007/s11090-019-10050-5.[6] R. Mehrabifard, Z. Kabarkouhi, F. Rezaei, K. Hajisharifi, H. Mehdian, and E. Robert, “Physical understanding of the static magnetic field’s synergistic enhancement of cold atmospheric pressure plasma treatment,” Apr. 2023, Accessed: Sep. 21, 2023. [Online]. Available: https://arxiv.org/abs/2304.05833v1 Figure 1

Fe-Fe Double-Atom Catalysts for Murine Coronavirus Disinfection: Nonradical Activation of Peroxides and Mechanisms of Virus Inactivation.

Peroxides find broad applications for disinfecting environmental pathogens particularly in the COVID-19 pandemic; however, the extensive use of chemical disinfectants can threaten human health and ecosystems. To achieve robust and sustainable disinfection with minimal adverse impacts, we developed Fe single-atom and Fe-Fe double-atom catalysts for activating peroxymonosulfate (PMS). The Fe-Fe double-atom catalyst supported on sulfur-doped graphitic carbon nitride outperformed other catalysts for oxidation, and it activated PMS likely through a nonradical route of catalyst-mediated electron transfer. This Fe-Fe double-atom catalyst enhanced PMS disinfection kinetics for inactivating murine coronaviruses (i.e., murine hepatitis virus strain A59 (MHV-A59)) by 2.17-4.60 times when compared to PMS treatment alone in diverse environmental media including simulated saliva and freshwater. The molecular-level mechanism of MHV-A59 inactivation was also elucidated. Fe-Fe double-atom catalysis promoted the damage of not only viral proteins and genomes but also internalization, a key step of virus lifecycle in host cells, for enhancing the potency of PMS disinfection. For the first time, our study advances double-atom catalysis for environmental pathogen control and provides fundamental insights of murine coronavirus disinfection. Our work paves a new avenue of leveraging advanced materials for improving disinfection, sanitation, and hygiene practices and protecting public health.

Susceptibility of enveloped and non-enveloped viruses to ultraviolet light-emitting diode (UV-LED) irradiation and implications for virus inactivation mechanisms

UV-LED treatment at 265 nm was more efficient than UV-LED treatment at 280 nm in inactivating both enveloped and non-enveloped viruses. Capsid damage is not important in virus inactivation by UV-LEDs for both enveloped and non-enveloped viruses.

Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model.

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

Review on inactivation of airborne viruses using non-thermal plasma technologies: from MS2 to coronavirus.

Although several non-thermal plasmas (NTPs) technologies have been widely investigated in air treatment, very few studies have focused on the inactivation mechanism of viruses by NTPs. Due to its efficiency and environmental compatibility, non-thermal plasma could be considered a promising virus-inactivation technology. Plasma is a partly or fully ionized gas including some species (i.e., electrons, free radicals, ions, and neutral molecules) to oxidize pollutants or inactivate harmful organisms. Non-thermal plasmas are made using less energy and have an active electron at a much higher temperature than bulk gas molecules. This review describes NTPs for virus inactivation in indoor air. The different application processes of plasma for microorganism inactivation at both laboratory and pilot-scale was also reviewed This paper reports on recent advances in this exciting area of viral inactivation identifying applications and mechanisms of inactivation, and summarizing the results of the latest experiments in the literature. Moreover, special attention was paid to the mechanism of virus inactivation. Finally, the paper suggests research directions in the field of airborne virus inactivation using non-thermal plasma.

Viral Inactivation Using Visible to UV Upconversion Nanomaterials and Application in Self-Cleaning Anti-Viral Nanofilms on Personal Protective Equipment (PPE) to Mitigate Coronavirus Spread

COVID-19 has become one of the deadliest pandemics in human history and has taken a tremendous toll on human life in large part due to its ability to transmit through the air and from surface to surface. We developed a novel conformal nano-coating on PPE surfaces intended to capture and kill (inactivate) COVID-19 viruses on PPE surfaces. Disinfection and Sterilization has long been done using UV radiation. Taking inspiration from this conventional method, we came up with a product that can produce UV radiation locally and regeneratively, a visible to UV Upconversion Nanomaterial (UCNP). The idea here is to expose viruses to localized UV radiation (within the mutagenic range) to kill or deactivate them. In our work, we developed a Yttrium Orthosilicate-based UCNP. These nanoparticles were coated on PPE surfaces and cationic and anionic polymers using the layer by layer (LBL) coating method. An oligosaccharide species, chosen to possess strong bond affinity/complementarity to novel coronavirus S glycoprotein, was grafted to the polymer using functional groups/sites via click chemistry to add one more mechanism of virus inactivation. In our testing, two formulations, Y2SiO5(1% Pr, 1% Gd) and Y2SiO5 (1.2% Pr, 1.2% Gd, 7.2% Li) have reduced active coronavirus viral load 4*104 to 0 in an hour of exposure to white light.

Characteristics and Mechanisms of Bacteriophage MS2 Inactivation in Water by UV Activated Sodium Persulfate

Viruses in the aquatic environment have strong resistance to common disinfection techniques. To contribute to the development of efficient virus inactivation technologies, the characteristics and mechanisms of virus inactivation in a UV activated sodium persulfate(UV/PS) system were studied. The inactivation rate and kinetic characteristics of bacteriophage MS2 in water samples by the UV/PS were studied. The effects of PS dosage, pH, and the initial concentration of bacteriophages on the inactivation effect were also investigated. Furthermore, the morphologies of phages before and after UV/PS treatment were observed by transmission scanning electron microscope, and the free radicals in the reaction system were identified by electron paramagnetic resonance spectroscopy. By means of a free radical quenching experiment, the contribution rate of various factors in the UV/PS system to phage inactivation was also analyzed and calculated. The results showed that when the UV irradiation intensity was 160 μW·cm-2, the phage MS2 of 4.39 lg could be removed after UV/PS treatment for 4 min, which was 1.44 lg higher than that of the same UV dose alone. The inactivation of phage MS2 by the UV/PS system was in accordance with the first-order kinetic model. Increasing the initial concentration of PS in the system significantly improved the inactivation rate of phages, while pH and the initial concentration of phages had little effect on the inactivation rate. UV/PS treatment damages the capsid of phages and promotes the aggregation of phage particles. The presence of SO4-· and·OH in the UV/PS system was an important factor for the inactivation of MS2 phages. Finally, ·OH contributed more to MS2 phage inactivation than SO4-·.

Mechanisms of instantaneous inactivation of SARS-CoV-2 by silicon nitride bioceramic.

The hydrolytic processes occurring at the surface of silicon nitride (Si3N4) bioceramic have been indicated as a powerful pathway to instantaneous inactivation of SARS-CoV-2 virus. However, the virus inactivation mechanisms promoted by Si3N4 remain yet to be elucidated. In this study, we provide evidence of the instantaneous damage incurred on the SARS-CoV-2 virus upon contact with Si3N4. We also emphasize the safety characteristics of Si3N4 for mammalian cells. Contact between the virions and micrometric Si3N4 particles immediately targeted a variety of viral molecules by inducing post-translational oxidative modifications of S-containing amino acids, nitration of the tyrosine residue in the spike receptor binding domain, and oxidation of RNA purines to form formamidopyrimidine. This structural damage in turn led to a reshuffling of the protein secondary structure. These clear fingerprints of viral structure modifications were linked to inhibition of viral functionality and infectivity. This study validates the notion that Si3N4 bioceramic is a safe and effective antiviral compound; and a primary antiviral candidate to replace the toxic and allergenic compounds presently used in contact with the human body and in long-term environmental sanitation.

Virus Inactivation in Water Using Laser-Induced Graphene Filters.

Interest in the pathogenesis, detection, and prevention of viral infections has increased broadly in many fields of research over the past year. The development of water treatment technology to combat viral infection by inactivation or disinfection might play a key role in infection prevention in places where drinking water sources are biologically contaminated. Laser-induced graphene (LIG) has antimicrobial and antifouling surface effects mainly because of its electrochemical properties and texture, and LIG-based water filters have been used for the inactivation of bacteria. However, the antiviral activity of LIG-based filters has not yet been explored. Here we show that LIG filters also have antiviral effects by applying electrical potential during filtration of the model prototypic poxvirus Vaccinia lister. This antiviral activity of the LIG filters was compared with its antibacterial activity, which showed that higher voltages were required for the inactivation of viruses compared to that of bacteria. The generation of reactive oxygen species, along with surface electrical effects, played a role in the mechanism of virus inactivation. This new property of LIG highlights its potential for use in water and wastewater treatment for the electrochemical disinfection of various pathogenic microorganisms, including bacteria and viruses.

Physiochemical properties of enveloped viruses and arginine dictate inactivation.

Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.

Kinetics and Mechanisms of Virus Inactivation by Chlorine Dioxide in Water Treatment: A Review.

Chlorine dioxide (ClO2), an alternative disinfectant to chlorine, has been widely applied in water and wastewater disinfection. This paper aims at presenting an overview of the inactivation kinetics and mechanisms of ClO2 with viruses. The inactivation efficiencies vary greatly among different virus species. The inactivation rates for different serotypes within a family of viruses can differ by over 284%. Generally, to achieve a 4-log removal, the exposure doses, also being referred to as Ct values (mutiplying the concentration of ClO2 and contact time) vary in the range of 0.06–10 mg L−1 min. Inactivation kinetics of viruses show two phases: an initial rapid inactivation phase followed by a tailing phase. Inactivation rates of viruses increase as pH or temperature increases, but show different trends with increasing concentrations of dissolved organic matter (DOM). Both damages in viral proteins and in the 5′ noncoding region within the genome contribute to virus inactivation upon ClO2 disinfection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00128-021-03137-3.

CHALLENGES OF TIO2-COMPOSITE, AS A VISIBLE ACTIVE PHOTOCATALYST MATERIAL FOR SARS-COV-2 ANTIVIRAL COMPARED WITH THE OTHER VIRUSES

The efforts in dealing with COVID-19 in various aspects are continuously being carried out, such as the photocatalytic process. Interestingly, TiO2 plays an important role as an environmentally friendly photocatalyst with a visible and UV absorption range. The increase in the antiviral performance of this compound is further maximized by formulating TiO2-based composites and doping it to reduce the bandgap. This review aims to determine the challenges of TiO2-based composite as a visible active photocatalyst material for SARS-CoV-2 antiviral. It presents a discussion of the photocatalytic function of TiO2, by comparing the secondary data obtained from viruses that have been successfully degraded by the SARS-CoV-2. The literature search led to the analysis of the photocatalyst quality, structure, composition, and the degraded viruses. This comparison was based on the photocatalyst’s general constituents, such as protein and RNA. The virus morphology as observed in the secondary data was successfully destroyed by hydroxyl radicals resulting from the photocatalytic processes. It was observed that TiO2 photocatalyst generated ROS, capable of inactivating various viruses, therefore, it has the potential in fighting against the SARS-CoV-2. Moreover, further research is needed in photocatalyst design, its role, and the overall mechanism of virus inactivation.

The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus under Conditions Relevant for Vegetable Sanitation.

We determined the disinfection efficacy and inactivation mechanisms of peracetic acid (PAA)-based sanitizer using pH values relevant for vegetable sanitation against rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). TV was significantly more resistant to PAA disinfection than RV: for a 2-log10 reduction of virus titer, RV required 1 mg/liter PAA for 3.5 min of exposure, while TV required 10 mg/liter PAA for 30 min. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanisms of virus inactivation were explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative PCR (RT-qPCR) and (ii) virus-host receptor interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). We observed that PAA induced damage to both RV and TV genomes and also decreased virus-receptor interactions, with the latter suggesting that PAA damages viral proteins important for binding its host cell receptors. Importantly, the levels of genome-versus-protein damage induced by PAA were different for each virus. PAA inactivation correlated with higher levels of RV genome damage than of RV-receptor interactions. For PAA-treated TV, the opposite trends were observed. Thus, PAA inactivates each of these viruses via different molecular mechanisms. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease.IMPORTANCE In this study, we examined the inactivation mechanisms of peracetic acid (PAA), a sanitizer commonly used for postharvest vegetable washing, for two enteric viruses: Tulane virus (TV) as a human norovirus surrogate and rotavirus (RV). PAA disinfection mechanisms for RV were mainly due to genome damage. In contrast, PAA disinfection in TV was due to damage of the proteins important for binding to its host receptor. We also observed that PAA triggered aggregation of TV to a much greater extent than RV. These studies demonstrate that different viruses are inactivated via different PAA mechanisms. This information is important for designing an optimal sanitation practice for postharvest vegetable washing to minimize foodborne viral diseases.

Water and Pathogenic Viruses Inactivation—Food Engineering Perspectives

Water is an essential component of food structures and biological materials. The importance of water as a parameter affecting virion stability and inactivation has been recognized across disciplinary areas. The large number of virus species, differences in spreading, likelihood of foodborne infections, unknown infective doses, and difficulties of infective virus quantification are often limiting experimental approaches to establish accurate data required for detailed understanding of virions’ stability and inactivation kinetics in various foods. Furthermore, non-foodborne viruses, as shown by the SARS-CoV-2 (Covid-19) pandemic, may spread within the food chain. Traditional food engineering benefits from kinetic data on effects of relative humidity (RH) and temperature on virion inactivation. The stability of enteric viruses, human norovirus (HuNoV), and hepatitis A (HAV) virions in food materials and their resistance against inactivation in traditional food processing and preservation is well recognized. It appears that temperature-dependence of virus inactivation is less affected by virus strains than differences in temperature and RH sensitivity of individual virus species. Pathogenic viruses are stable at low temperatures typical of food storage conditions. A significant change in activation energy above typical protein denaturation temperatures suggests a rapid inactivation of virions. Furthermore, virus inactivation mechanisms seem to vary according to temperature. Although little is known on the effects of water on virions’ resistance during food processing and storage, dehydration, low RH conditions, and freezing stabilize virions. Enveloped virions tend to have a high stability at low RH, but low temperature and high RH may also stabilize such virions on metal and other surfaces for several days. Food engineering has contributed to significant developments in stabilization of nutrients, flavors, and sensitive components in food materials which provides a knowledge base for development of technologies to inactivate virions in foods and environment. Novel food processing, particularly high pressure processing (HPP) and cold plasma technologies, seem to provide efficient means for virion inactivation and food quality retention prior to packaging or food preservation by traditional technologies.

Assessing the Occurrence of Waterborne Viruses in Reuse Systems: Analytical Limits and Needs.

Detection of waterborne enteric viruses is an essential tool in assessing the risk of waterborne transmission. Cell culture is considered a gold standard for detection of these viruses. However, it is important to recognize the uncertainty and limitations of enteric virus detection in cell culture. Cell culture cannot support replication of all virus types and strains, and numerous factors control the efficacy of specific virus detection assays, including chemical additives, cell culture passage number, and sequential passage of a sample in cell culture. These factors can result in a 2- to 100-fold underestimation of virus infectivity. Molecular methods reduce the time for detection of viruses and are useful for detection of those that do not produce cytopathogenic effects. The usefulness of polymerase chain reaction (PCR) to access virus infectivity has been demonstrated for only a limited number of enteric viruses and is limited by an understanding of the mechanism of virus inactivation. All of these issues are important to consider when assessing waterborne infectious viruses and expected goals on virus reductions needed for recycled water. The use of safety factors to account for this may be useful to ensure that the risks in drinking water and recycled water for potable reuse are minimized.

Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes.

Virus inactivation mechanisms can be elucidated by methods that measure the loss of specific virus functionality (e.g., host attachment, genome internalization, and genome replication). Genome functionality is frequently assessed by PCR-based methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we developed here a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV light at 254 nm (UV254), or heat, and then the reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV254, genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) after disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection.IMPORTANCE This study provides a new tool for assessing virus inactivation mechanisms by directly measuring a viral genome's ability to produce new viruses after disinfection. In addition, we identify a potential pitfall of PCR for determining virus genome damage, which does not reflect whether a genome is truly functional. The results presented here using quantitative transfection corroborate previously suggested virus inactivation mechanisms for some virus inactivation methods (heat) while bringing additional insights for others (ozone, FC, and UV254). The developed transfection method provides a more mechanistic approach for the assessment of actual virus inactivation by common water disinfectants.

Mechanism of Virus Inactivation by Cold Atmospheric-Pressure Plasma and Plasma-Activated Water

Viruses cause serious pathogenic contamination that severely affects the environment and human health. Cold atmospheric-pressure plasma efficiently inactivates pathogenic bacteria; however, the mechanism of virus inactivation by plasma is not fully understood. In this study, surface plasma in argon mixed with 1% air and plasma-activated water was used to treat water containing bacteriophages. Both agents efficiently inactivated bacteriophages T4, Φ174, and MS2 in a time-dependent manner. Prolonged storage had marginal effects on the antiviral activity of plasma-activated water. DNA and protein analysis revealed that the reactive species generated by plasma damaged both nucleic acids and proteins, consistent with the morphological examination showing that plasma treatment caused the aggregation of bacteriophages. The inactivation of bacteriophages was alleviated by the singlet oxygen scavengers, demonstrating that singlet oxygen played a primary role in this process. Our findings provide a potentially effective disinfecting strategy to combat the environmental viruses using cold atmospheric-pressure plasma and plasma-activated water.IMPORTANCE Contamination with pathogenic and infectious viruses severely threatens human health and animal husbandry. Current methods for disinfection have different disadvantages, such as inconvenience and contamination of disinfection by-products (e.g., chlorine disinfection). In this study, atmospheric surface plasma in argon mixed with air and plasma-activated water was found to efficiently inactivate bacteriophages, and plasma-activated water still had strong antiviral activity after prolonged storage. Furthermore, it was shown that bacteriophage inactivation was associated with damage to nucleic acids and proteins by singlet oxygen. An understanding of the biological effects of plasma-based treatment is useful to inform the development of plasma into a novel disinfecting strategy with convenience and no by-product.

Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in Response to a Simultaneous Application of UV and Chlorine.

The simultaneous application of UV and chlorine (expressed as UV/Cl2) as a water treatment method may be a good disinfection option for UV-resistant microorganisms, such as human adenoviruses (HAdVs). In this study, we developed two approaches using UV/Cl2: one to quantitate the OH• radicals based on the degradation of the probe compound para-chlorobenzoic acid (pCBA) and the other to use bacteriophage MS2 to understand the virus inactivation mechanisms in response to UV, chlorine and UV/Cl2 disinfection using reverse-transcription quantitative polymerase chain reaction (RT-qPCR), attachment and genome penetration assays. The results revealed that OH• radicals were produced at a concentration of 2.70 × 10-14 M in the UV/Cl2 treatment with a practical chlorine dose of 1 mg/L and with a minimum UV254 fluence of approximately 10 mJ/cm2, whereas UV or chlorine alone did not produce OH• radicals. In the UV/Cl2 treatment, synergistic effects on viral genome damage were observed, but were not directly due to OH• radicals. The ability of MS2 to penetrate the genome of the host bacteria was impaired, but its ability to attach to the host was not affected by the treatment. We concluded that the major cause of virus inactivation in response to UV/Cl2 was the damage to the viral genome caused by combination actions of chlorine species and OH• radicals.