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Among the reported methods, the ozone treatment seems very promising 4 , 5 , 6 , 7. Ozone is the most powerful oxidant found in nature 6 , 7 , 8 , and due to its instability-it decomposes by itself- the ozonization process only needs a short exposure time, whereby there are no harmful residuals that need to be removed after ozonization processes 8 , 9.

The potential usefulness and benefit of the ozonization disinfecting process ODP have already been reported 9 , 10 , 11 , Although the beneficial effects of Ozone treatment against many viruses have been reported since the s, the inactivation mechanism of action is still relatively unknown Some studies have reported the effect of ozone on cysteines in the surface proteins of coronaviruses, in which ozone and other oxidants can inhibit virus entry into host cells The aim of this study was to investigate the effect of ozone on this virus and to find the mechanisms of its effect so that ozone can be used as a disinfectant in different places in current epidemic conditions.

Also, possible mechanisms of the effect of ozone on viruses have been expressed in a scattered manner in previous studies, so this study also explains several mechanisms together. Also, a 3D image of this particle is shown in Fig. Figure 2 e,f show the height and phase images of some ozone-treated virus particles in a microscopic field.

Also, Transmission electron microscopy showed the round shape of a damaged virus particle with an average size of 80— nm Fig. The exposure time in Fig. Coronaviruses, especially the SARS-CoV-2, are rich in cysteine, and their residues must be intact for viral activity, mainly through zinc ion participation 14 , It is known that conserved cysteine residues are important in virus stability and the loss of more than one cysteine affects the formation of the viral envelope Desorption of proteins occurs from the boundaries of the membrane patches, gradually leading to the release of proteins.

As it is observed in both lateral phase images Fig. Kuznetsov et al. Further, ozone attacks the unsaturated fatty acids in the membrane with the possible consequence of lipid peroxidation 9 , which is observed by increased malondialdehyde, one of the aldehydic products of lipid peroxidation Another slight blue shift is evident in the IR-spectra the right spectra from The intensity of these modes shows that the phospholipid layers are cramped, packed, and torn.

Ozone exposure results in protein secondary and tertiary structure destruction 9. It has been observed that ozone attacks the amino acids directly, particularly cysteine, methionine, tryptophan, tyrosine, and histidine 9. Upon exposure to ozone, cysteine is oxidized as the first step to cystine, but further oxidation by ozone converts cysteine to cystic acid 8 , Due to the important role of conserved cysteine residues, the conversion of cysteine to cystine was monitored over 20 min of exposure to ozone Supplementary Information, Figure S2.

As observed in Supplementary Information, Figure S3 , the intensity of the SH bond decreased dramatically, probably because of cysteine disulfide bond formation. The viral suspension samples were also exposed to ozone and were then evaluated by transmission electron microscopy and AFM techniques. The TEM image, Supplementary Information, Figure S4 , shows two connected virus particles; it seems SH groups from adjacent viruses are linked to form disulfide bonds after 2 min of ozone exposure.

After 2 min, they bind together due to the formation of a disulfide bond as it is shown in Fig. The effect of ozone on the morphology of the virus at different exposure times 2, 4, and 6 min has also been investigated, as shown in Fig.

It is observed that cysteine residue SH groups readily oxide to disulfide upon ozone exposure. AFM images of the virus aggregation at 2 min ozone exposure time; a 2D image of aggregated virus particles. AFM images showing the effect of ozone on the virus at different times 2, 4, and 6 min in a , b , and c , respectively. After 2 min of exposure to ozone 2D, lateral, and 3D views , d , e , and f , respectively.

After 4 min of exposure to ozone 2D, lateral, and 3D views , g , h , and i , respectively. After 6 min of exposure to ozone 2D, lateral, and 3D views. As it is already mentioned, the virus cysteine residues need to be intact in the virus protein structures, mainly through zinc ion participation, to be able to play their crucial role in the viral activity 14 , Zinc ions participate in many different cellular processes, and zinc-cysteine finger motifs have been proven to play a crucial role in proper protein-folding, catalysis, regulation of various enzymes, transcription factors, and other activities still under study.

A group of these finger domains that stabilizes protein structures can interact with other biomolecules, such as RNA and DNA. Anja Seybert et al. Atomic absorption spectroscopy was applied to measure the concentration of zinc in the supernatant after being exposed to ozone for 2 min. The zinc concentration was found to be approximately one part per million.

It was observed that the attachment of the zinc-cysteine complex to the silicon surface is lost upon the release of zinc from cysteine moiety as a result of ozone exposure Supplementary Information, Figure S3.

Zinc ions participate in many different cellular processes, and zinc-cysteine finger domains have been proven to have a crucial role in the proper folding, catalysis, and regulations of various enzymes and transcription factors 23 , Coronaviruses employ a set of non-structural proteins nsp produced as cleavage products of the ORF1a and ORF1ab viral polyproteins that assemble to assist viral replication and transcription Two types of zinc finger motifs structurally stabilize nsp3 for interaction with other proteins and biomolecules Fig.

According to Fig. It has been shown that the nsp14—nsp10 complex is involved in RNA viral proofreading 30 , 31 , Accordingly, nsp10 is considered a great target to develop broad-spectrum inhibitors for repressing coronaviruses activity Nsp10 includes two zinc finger motifs Fig.

The first Fig. Studies have shown that mutation in Zn coordinated residues abrogate nsp10 interactions with nsp14 and nsp16, probably due to the protein misfolding It should also be mentioned that SARS Coronavirus nsp14 includes three Zn finger motifs from which the one in the proximity of the active site plays a role in the catalysis, and the other two are responsible for the enzyme structure stability 25 , Computational studies suggest that the zinc motifs are a part of a conserved mechanism of RdRp switching, which changes RdRp domain orientations to stabilize the protein structure in different phases of its catalytic activity The effect of ozone exposure on the virus was monitored at different times Fig.

At the exposure time of 2 min, the viruses are damaged and destroyed. As can be seen in Fig. It seems the viruses that have lost their genomes appear narrower in the 3D image because of the finite size of the AFM tip. Increasing the exposure time results in the formation of new crystals, probably from the remnants of the damaged virus particles. AFM images showed significant heterogeneity on the surface of viruses after ozone treatment.

The results of atomic absorption spectroscopy and transmission electron microscopy showed that ozone breaks down virus proteins through three mechanisms; it destroys the virus envelope, converts cysteine to cystine through disulfide bond formation between adjacent virus particles, and finally release Zn ions from the virus protein structures which eventually destroy the structure of the proteins and alter their proper activity.

In addition, increasing the time of ozone exposure leads to the formation of new crystals, which may be damaged by the remnants of virus particles. The obtained results would definitely expand our knowledge to develop virus elimination techniques as well as potential therapeutics and treatment mechanisms employing oxidizing agents like ozone.

MilliQ ultrapure water Patients spike samples were considered for the study if they had given written informed consent and had clinical evidence of COVID confirmed by a PCR test.

The work was conducted in compliance with principles of the Declaration of Helsinki and is also approved by the Iran University of Medical Sciences Ethics Committee with an ethics code of IR. Nasopharyngeal and oropharyngeal swab and sputum samples were collected from symptomatic patients to detect SARS-CoV-2 by real-time reverse transcriptase assay.

Subsequently, the carrier RNA-containing lysis buffer was added to the tube and mixed by pulse-vortexing. Ethanol was added to the tube and incubated for 5 min at room temperature. The lysate was applied into columns connected to a vacuum system. After performing the washing steps, the tube was centrifuged at full speed for 1 min to completely dry the membrane.

Twenty microliters of the sample were added to each tube. Positive control, negative control, and no-template control NTC were also included in the test. Forty samples, which had higher viral loads, were pooled and then divided into different containers and exposed to ozone for different times from 2 to 6 min. The silicon wafer and ATR crystal were prepared according to a study by Alavi et al.

Trace amounts of organic containment were removed using Piranha solution The obtained H-Si surface was then washed with deoxygenated MilliQ water, dried, and immediately placed into a quartz Schlenk tube containing 20 ml of deoxygenated neat undecanoic acid UD , then purged with argon for 30 min, and finally, exposed to UV illumination nm, Black-Ray B AP Lamp, W 4 h After weight precipitation appeared, the sample was filtered and dried and the correspondence spectrum was graphed by diamond ATR.

AFM Imaging was performed in the attractive force regime, in which the tip is in very weak interaction with the surface or low drive amplitude. In the negative staining process, a drop of the viral suspension was placed immediately on the carbon-coated copper grid and left for 3—5 min.

Determination of zinc after centrifugation in the supernatant was carried out by atomic absorption spectroscopy, using a GF-AAS instrument 3. The measurements were made employing an air acetylene flame with a spectral groove width of 0. The solutions for the construction of the calibration curve were made from a standard solution Merck KGaA brand copper standard ppm. Viral transport media was used as a blank in this procedure. An average of — scans was used to collect data for each spectrum, and liquid nitrogen-cooled mercury cadmium telluride MCTA was utilized as a detector.

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SARS-coronavirus spike S2 domain flanked by cysteine residues C and C is important for activation of membrane fusion.

Virology , — Kuznetsov, Y. Atomic force microscopy investigation of HIV-infected lymphocytes atomic force microscopy ininfected lymphocytes. Ayala, A. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxynonenal. Cell Longev. Frey, S. Orientation of melittin in phospholipid bilayers. Nov 24th, Not a member of Pastebin yet?

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