pH-dependent endocytosis mechanisms for influenza A and SARS-coronavirus.

The ongoing SARS-CoV-2 pandemic and the influenza epidemics have revived the interest in understanding how these highly contagious enveloped viruses respond to alterations in the physicochemical properties of their microenvironment. By understanding the mechanisms and conditions by which viruses exploit the pH environment of the host cell during endocytosis, we can gain a better understanding of how they respond to pH-regulated anti-viral therapies but also pH-induced changes in extracellular environments. This review provides a detailed explanation of the pH-dependent viral structural changes preceding and initiating viral disassembly during endocytosis for influenza A (IAV) and SARS coronaviruses. Drawing upon extensive literature from the last few decades and latest research, I analyze and compare the circumstances in which IAV and SARS-coronavirus can undertake endocytotic pathways that are pH-dependent. While there are similarities in the pH-regulated patterns leading to fusion, the mechanisms and pH activation differ. In terms of fusion activity, the measured activation pH values for IAV, across all subtypes and species, vary between approximately 5.0 to 6.0, while SARS-coronavirus necessitates a lower pH of 6.0 or less. The main difference between the pH-dependent endocytic pathways is that the SARS-coronavirus, unlike IAV, require the presence of specific pH-sensitive enzymes (cathepsin L) during endosomal transport. Conversely, the conformational changes in the IAV virus under acidic conditions in endosomes occur due to the specific envelope glycoprotein residues and envelope protein ion channels (viroporins) getting protonated by H+ ions. Despite extensive research over several decades, comprehending the pH-triggered conformational alterations of viruses still poses a significant challenge. The precise mechanisms of protonation mechanisms of certain during endosomal transport for both viruses remain incompletely understood. In absence of evidence, further research is needed.

UV radiation sensitivity of bacteriophage PhiX174- A potential surrogate for SARS-CoV-2 in terms of radiation inactivation

To minimize health risks, surrogates are often employed to reduce experiments with pathogenic microorganisms and the associated health risk. Due to structural similarities between the enveloped RNA -viruses SARS-CoV-2 and Phi6, the latter has been established as a nonpathogenic coronavirus surrogate for many applications. However, large discrepancies in the UV log-reduction doses between SARS-CoV-2 and Phi6 necessitate the search for a better surrogate for UV inactivation applications. A literature study provided the bacteriophage PhiX174 as a potentially more suitable nonpathogenic coronavirus surrogate candidate. In irradiation experiments, the sensitivity of PhiX174 was investigated upon exposure to UV radiation of wavelengths 222 nm (Far-UVC), 254 nm (UVC), 302 nm (broad-band UVB), 311 nm (narrow-band UVB) and 366 nm (UVA) using a plaque assay. The determined log-reduction doses for PhiX174 were 1.3 mJ/cm2 @ 222 nm, 5 mJ/cm2 @ 254 nm, 17.9 mJ/cm2 @ 302 nm, 625 mJ/cm2 @ 311 nm and 42.5 J/cm2 @ 366 nm. The comparison of these results with published log-reduction doses of SARS-CoV-2 in the same spectral region, led to the conclusion that the bacteriophage PhiX174 exhibits larger log-reduction doses than SARS-CoV-2, nevertheless, it is a better UV-surrogate at 222 nm (Far-UVC), 254 nm (UVC) and 302 nm (UVB) than the often applied Phi6.

Identification of small molecules capable of enhancing viral membrane fusion.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

A review of the antiviral activity of cationic antimicrobial peptides.

Viral epidemics are occurring frequently, and the COVID-19 viral pandemic has resulted in at least 6.5 million deaths worldwide. Although antiviral therapeutics are available, these may not have sufficient effect. The emergence of resistant or novel viruses requires new therapies. Cationic antimicrobial peptides are agents of the innate immune system that may offer a promising solution to viral infections. These peptides are gaining attention as possible therapies for viral infections or for use as prophylactic agents to prevent viral spread. This narrative review examines antiviral peptides, their structural features, and mechanism of activity. A total of 156 cationic antiviral peptides were examined for information of their mechanism of action against both enveloped and non-enveloped viruses. Antiviral peptides can be isolated from various natural sources or can be generated synthetically. The latter tend to be more specific and effective and can be made to have a broad spectrum of activity with minimal side effects. Their unique properties of being positively charged and amphipathic enable their main mode of action which is to target and disrupt viral lipid envelopes, thereby inhibiting viral entry and replication. This review offers a comprehensive summary of the current understanding of antiviral peptides, which could potentially aid in the design and creation of novel antiviral medications.

Circulating pediatric respiratory pathogens in Taiwan during 2020: Dynamic change under low COVID-19 incidence.

BACKGROUND: In Taiwan, there were only 799 confirmed COVID-19 cases in 2020. The unique backdrop amidst a pandemic and promotion of nonpharmaceutical interventions generated some distinct changes in the epidemiology of common respiratory pathogens. In this study, we aimed to investigate the dynamic changes in respiratory pathogens in children during 2020. METHODS: We performed a retrospective cohort study at a tertiary hospital in southern Taiwan during 2020. Patients aged 0-18 years who visited the pediatric emergency department were enrolled. Children who presented with clinical symptoms (fever or respiratory illness) and received nasopharyngeal swabs for multiplex polymerase chain reaction (PCR) were included in our analysis. We also compared respiratory syncytial virus (RSV) trends from previous years by PCR and lateral flow immunochromatographic assays from 2017 to 2020. RESULTS: A total of 120 children were tested. The overall detection rate was 55%. With strengthened restrictions, the detection rate dropped from 70% to 30%. However, non-enveloped viruses (rhinovirus/enterovirus and adenovirus) were in constant circulation. Upon easing prevention measures, the detection rate remained above 60%, and an outbreak of an enveloped virus (RSV and parainfluenza virus) was noted. Compared with 2017-2019, the cyclical RSV epidemic was delayed, with a large surge in late 2020. CONCLUSIONS: We observed a constant circulation of non-enveloped viruses when strict nonpharmaceutical interventions were employed and a delayed surge of enveloped viruses during the easing of restrictions. Continuous surveillance and monitoring of the evolutionary dynamics of respiratory viruses is important, while easing restrictions requires balanced judgment.

Characterization of extracellular vesicle and virus-like particles by single vesicle tetraspanin analysis

Extracellular vesicles (EVs) are nano-sized membranous particles secreted by cells. EVs have been classified into subpopulations according to their presumed biogenesis pathway, but their detailed biogenesis mechanisms still need to be fully elucidated. Enveloped viruses are another type of cell-derived nano-vesicles, and their biogenesis processes are much better known than that of EVs. Recently, studies on the similarity between enveloped viruses and EVs have been increasingly reported. The biogenesis of EVs could be better understood if these similarities are adequately investigated. In this study, we utilized a single vesicle imaging technique to visualize the protein expressions of individual nano-sized vesicles and analyzed expression patterns within single vesicles. Using this technique, we identified unique tetraspanin expression patterns in single EVs and that these patterns were closely related to their subcellular origins. The expression of CD9 or CD81 in EVs implied that they originated from the plasma membrane, and the expression of CD63 in EVs implied that they originated from endosomal organelles. We further analyzed the tetraspanin expressions of two different types of virus-like particles (VLPs) and demonstrated that the HIV-Gag-induced VLPs were more similar to EVs than SARS-CoV-2-NP/M/E-induced VLPs. In addition, HIV-Gag-GFP-expressing VLPs were highly colocalized with CD9, CD63, and CD81 signals, whereas SARS-CoV-NP-GFP-expressing VLPs were not. Based on these observations, we could assume that tetraspanin-expressing EVs might be produced through a similar process by which HIV is produced. © 2023

Interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity.

Infections caused by enveloped viruses require fusion with cellular membranes for viral genome entry. Viral entry occurs following an interaction of viral and cellular membranes allowing the formation of fusion pores, by which the virus accesses the cytoplasm. Here, we focus on interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity. IFITM3 is predicted to block or stall viral fusion at an intermediate state, causing viral propagation to fail. After introducing IFITM3, we describe the generalized lipid membrane fusion pathway and how it can be stalled, particularly with respect to IFITM3, and current questions regarding IFITM3's topology, with specific emphasis on IFITM3's amphipathic α-helix (AAH) 59V-68M, which is necessary for the antiviral activity. We report new hydrophobicity and hydrophobic moment calculations for this peptide and a variety of active site peptides from known membrane-remodeling proteins. Finally, we discuss the effects of posttranslational modifications and localization, how IFITM3's AAH may block viral fusion, and possible ramifications of membrane composition.

A novel antiviral formulation containing caprylic acid inhibits SARS-CoV-2 infection of a human bronchial epithelial cell model.

A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

Boronic acid-modified bacterial cellulose microspheres as packing materials for enveloped virus removal.

Viruses are the most abundant microorganisms on the earth, their existence in contaminated waters possesses a significant threat to humans. Waterborne viral infections could be fatal to sensitive population including young child, the elderly, and the immune-compromised. It is imperative to remove viruses during water treatment to better protect public health, especially in the light of evidence of detection of coronaviruses genetic fragments in raw sewage. We reported bench-scale experiments evaluating the extent and mechanisms of removal of a model virus (spring viremia of carp virus, SVCV) in water by adsorption. Microspheres made by boronic acid-modified bacterial cellulose with excellent mechanical strength were successfully fabricated as packing materials for the column to remove glycoproteins and enveloped viruses from water. The synthesized adsorbent was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), and Brunauer Emmett Teller (BET) measurement. The adsorption efficiency of glycoproteins was investigated by SDS-PAGE and the Broadford protein assay, while the binding capacity with the virus (spring viremia of carp virus) was monitored by cell culture to calculate the viral cytopathic effect and viral titer caused by the virus. The data obtained from the above experiments showed that ∼3-log removal of SVCV in 3 h, which significantly reduced the virus concentration from microspheres packed column. The present study provides substantial evidence to prove beyond doubt that material based on bacterial cellulose seems to have the potential for virus removal from water which can be extended to systems of significant importance.

Molecular dynamics simulation of interaction between nanorod and phospholipid molecules bilayer

Natural and artificially prepared nanorods' surfaces have proved to have good bactericidal effect and self-cleaning property. In order to investigate whether nanorods can kill the enveloped virus, like destroying bacterial cell, we study the interaction between nanorods and virus envelope by establishing the models of nanorods with different sizes as well as the planar membrane and vesicle under the Dry Martini force field of molecular dynamics simulation. The results show that owing to the van der Waals attraction between nanorods and the tail hydrocarbon chain groups of phospholipid molecules, the phospholipid molecules on virus envelope are adsorbed to nanorods on a large scale. This process will increase the surface tension of lipid membrane and reduce the order of lipid molecules, resulting in irreparable damage to planar lipid membrane. Nanorods with different diameters have different effects on vesicle envelope, the larger the diameter of nanorod, the weaker the van der Waals effect on the unit cross-sectional area is and the smaller the degree of vesicle deformation. There is synergy between the nanorods in the nanorod array, which can enhance the speed and scale of lipid adsorption. The vesicle adsorbed in the array are difficult to desorb, and even if desorbed, vesicle will be seriously damaged. The deformation rate of the vesicle adsorbed in the nanorod array exceeds 100%, implying that the nanorod array has a strong destructive effect on the vesicle. This preliminarily proves the feasibility of nanorod array on a surface against enveloped virus, and provides a reference for the design of corresponding nanorods surface.

Inhibition of Enveloped Virus Surrogate Phi6 Infection Using Yeast-Derived Vacuoles.

The periodic emergence of infectious disease poses a serious threat to human life. Among the causative agents, including pathogenic bacteria and fungi, enveloped viruses have caused global pandemics. In the last 10 years, outbreaks of severe acute respiratory syndrome coronavirus 2 disease, severe acute respiratory syndrome, and Middle East respiratory syndrome have all been caused by enveloped viruses. Among several paths of secondary transmission, inhalation of aerosols containing saliva with sputum droplets from infected patients is the major path. To prevent these infectious diseases, mass use of antiviral agents is essential. The yeast-derived vacuole is a small organelle in which hydrolytic enzymes are concentrated. It is an intracellular organ with an excellent ability to process old organelles and bacteria and viruses that have invaded from the outside and can be present in sufficient quantity to be called a kind of enzyme bomb. We confirmed the inhibition of virus infection and structural collapse by vacuole treatment. Among several enzymes, proteases affected Phi6 infectivity. This study tried to isolate these vacuoles from yeast and use them as an antiviral agent for virus treatment, which is a recent issue. We confirmed that viral infectivity was inactivated, and structure collapsed through vacuole treatment. This paper is meaningful in that extracellularly isolated yeast-derived vacuoles are a first attempt to utilize vacuoles for viral treatment. IMPORTANCE The study assesses the vacuoles isolated from the yeast Saccharomyces cerevisiae as green antiviral agents to decrease the concerns about massive use of chemical antiviral agents and its side effects. To prevent the spreading of infectious diseases, personal or public use of antiviral agents is encouraged. The concern about the active compounds of these chemical antiviral agents has grown. Active compounds of antiviral agents have potential side effects on human health and the environment. Our proposed approach suggests effective and green antivirus material from a nonhazardous yeast strain. Also, large-scale production using a fermentation process can allow cost-effectiveness. The results showed sufficient reduced infectivity by vacuole treatment. The exposed vacuole can play the roles of both enzyme bomb to the virus and renewable nutrient source in the ecosystem.

Photodynamic viral inactivation assisted by photosensitizers

The deadly viruses, which are spreading worldwide at an alarming rate, are a major challenge for the life sci-ences. More efficient and cost-effective methods with fewer side effects can provide a good alternative to traditional drug-based methods. Currently, physical phenomena such as light in the form of photodynamic action are increasingly being used to inactivate viruses. Photodynamic inactivation (PDI) uses a photosensitizer (PS), light, and oxygen to generate reactive oxygen species (ROS) to inactivate microorganisms. This article reviews the use of existing PSs, as one of the essential anti-viral agents, and introduces new materials and strategies combined with PDI. Physiochemical properties of PSs and their role in interaction with virus components are discussed. Furthermore, the effectiveness of optical sensitizers with radiation methods to inactivate viruses is highlighted.

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus.

BACKGROUND: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure). METHODS: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate. RESULTS: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces. DISCUSSION AND CONCLUSIONS: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Expanding the antiviral potential of the mosquito lipid-transfer protein AEG12 against SARS-CoV-2 using hydrophobic antiviral ligands.

The mosquito protein AEG12 encompasses a large (~ 3800 Å3 ) hydrophobic cavity which binds and delivers unsaturated fatty acids into biological membranes, allowing it to lyse cells and neutralize a wide range of enveloped viruses. Herein, the lytic and antiviral activities are modified with non-naturally occurring lipid ligands. We generated novel AEG12 complexes in which the endogenous fatty acid ligands were replaced with hydrophobic viral inhibitors. The resulting compounds modulated cytotoxicity and infectivity against SARS-CoV-2, potentially reflecting additional mechanisms of action beyond membrane destabilization. These studies provide valuable insight into the design of novel broad-spectrum antiviral therapeutics centred on the AEG12 protein scaffold as a delivery vehicle for hydrophobic therapeutic compounds.

Antiviral Activity and Mechanisms of Seaweeds Bioactive Compounds on Enveloped Viruses-A Review.

In the last decades, the interest in seaweed has significantly increased. Bioactive compounds from seaweed's currently receive major attention from pharmaceutical companies as they express several interesting biological activities which are beneficial for humans. The structural diversity of seaweed metabolites provides diverse biological activities which are expressed through diverse mechanisms of actions. This review mainly focuses on the antiviral activity of seaweed's extracts, highlighting the mechanisms of actions of some seaweed molecules against infection caused by different types of enveloped viruses: influenza, Lentivirus (HIV-1), Herpes viruses, and coronaviruses. Seaweed metabolites with antiviral properties can act trough different pathways by increasing the host's defense system or through targeting and blocking virus replication before it enters host cells. Several studies have already established the large antiviral spectrum of seaweed's bioactive compounds. Throughout this review, antiviral mechanisms and medical applications of seaweed's bioactive compounds are analyzed, suggesting seaweed's potential source of antiviral compounds for the formulation of novel and natural antiviral drugs.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

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.

The Analogs of Furanyl Methylidene Rhodanine Exhibit Broad-Spectrum Inhibitory and Inactivating Activities against Enveloped Viruses, including SARS-CoV-2 and Its Variants.

In recent years, infectious diseases caused by viral infections have seriously endangered human health, especially COVID-19, caused by SARS-CoV-2, which continues to spread worldwide. The development of broad-spectrum antiviral inhibitors is urgently needed. Here, we report a series of small-molecule compounds that proved effective against human coronaviruses (HCoV), such as SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), SARS-CoV, MERS-CoV, HCoV-OC43, and other viruses with class I viral fusion proteins, such as influenza virus, Ebola virus (EBOV), Nipah virus (NiV), and Lassa fever virus (LASV). They are also effective against class II enveloped viruses represented by ZIKV and class III enveloped viruses represented by vesicular stomatitis virus (VSV). Further studies have shown that these compounds may exert antiviral effects through a variety of mechanisms, including inhibiting the formation of the six-helix bundle, which is a typical feature of enveloped virus fusion with cell membranes, and/or targeting viral membrane to inactivate cell-free virions. These compounds are expected to become drug candidates against SARS-CoV-2 and other enveloped viruses.

Quantitative evaluation of the inactivation effect of ozonated water on SARS-CoV-2 based on corrected CT values

There are many issues in the evaluation protocols based on CT (mg min/L) values, which have been used to assess the germicidal effect of highly oxidative and unstable ozonated water. The major problems include the carryover of culture medium components in virus inactivation assays and the reaction volume ratio between the virus suspension and ozonated water. Furthermore, it is essential to correct the CT value with the decay curve of dissolved ozone under the same conditions as the inactivation assay. In this study, these concerns were reexamined to obtain quantitative CT values. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation test using ozonated water prepared from pure water was assessed by determining the corrected concentration time (CCT) values. Moreover, a possible inactivation mechanism of SARS-CoV-2 was discussed with the aid of findings from this study and previous reports. The findings revealed that the CCT value required for 99.95% inactivation of SARS-CoV-2 with ozonated water was 0.97 mg·min/L. To quantitatively evaluate the SARS-CoV-2 inactivation test, the virus purification procedure during the pretreatment and the CT value correction using a dissolved ozone decay curve obtained under the same condition as the inactivation test were demonstrated to be essential.We proposed a possible mechanism of SARS-CoV-2 inactivation with ozonated water. Amino acids such as tyrosine, tryptophan, methionine, cysteine, and histidine in the SARS-CoV-2 spike protein are susceptible to oxidative attack by the ozone dissolved in water. This attack may induce structural destruction of the spike protein and inhibit its binding to the angiotensin converting enzyme 2 (ACE2) receptor, an essential host receptor for viral infection, resulting in viral inactivation and contributing to infection suppression. [ FROM AUTHOR] Copyright of Ozone: Science & Engineering is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)