Inactivation of SARS-CoV-2 by a chitosan/α-Ag2WO4 composite generated by femtosecond laser irradiation

In the current COVID-19 pandemic, the next generation of innovative materials with enhanced anti-SARS-CoV-2 activity is urgently needed to prevent the spread of this virus within the community. Herein, we report the synthesis of chitosan/α-Ag2WO4 composites synthetized by femtosecond laser irradiation. The antimicrobial activity against Escherichia coli, Methicilin-susceptible Staphylococcus aureus (MSSA), and Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). To assess the biocompatibility of chitosan/α-Ag2WO4 composites in a range involving MIC and MBC/MFC on keratinocytes cells (NOK-si), an alamarBlue™ assay and an MTT assay were carried out. The SARS-CoV-2 virucidal effects was analyzed in Vero E6 cells through viral titer quantified in cell culture supernatant by PFU/mL assay. Our results showed a very similar antimicrobial activity of chitosan/α-Ag2WO4 3.3 and 6.6, with the last one demonstrating a slightly better action against MSSA. The chitosan/α-Ag2WO4 9.9 showed a wide range of antimicrobial activity (0.49–31.25 µg/mL). The cytotoxicity outcomes by alamarBlue™ revealed that the concentrations of interest (MIC and MBC/MFC) were considered non-cytotoxic to all composites after 72 h of exposure. The Chitosan/α-Ag2WO4 (CS6.6/α-Ag2WO4) composite reduced the SARS-CoV-2 viral titer quantification up to 80% of the controls. Then, our results suggest that these composites are highly efficient materials to kill bacteria (Escherichia coli, Methicillin-susceptible Staphylococcus aureus, and the yeast strain Candida albicans), in addition to inactivating SARS-CoV-2 by contact, through ROS production.


Results and discussion
Structural analysis. The α-Ag 2 WO 4 was synthesized by the coprecipitation method as previously reported [45][46][47] (see SI for experimental details). Figure S2A shows the X-ray diffraction (XRD) patterns of the α-Ag 2 WO 4 microcrystal, confirming its orthorhombic structure according to the inorganic crystal structure database (ICSD)-card no. 4165 48 . This figure also presents the XRD peaks related to the semi-crystalline structure of the CS polymer, showing intense peaks located at 11.4°, 21.0° and 22.5°. Figure S2B displays the XRD patterns of the CS/α-Ag 2 WO 4 composites irradiated by fs laser, where a characteristic XRD pattern of an amorphous phase related to the CS polymer and peaks assigned to the α-Ag 2 WO 4 orthorhombic structure can be observed. According to Khan et al. 49 , the crystallinity of the CS polymer is ascribed to the number of -NH 2 and -OH groups in the structure. These groups form strong intra and intermolecular hydrogen bonds, leading to a certain regularity of the CS structure, thus resulting in the appearance of crystalline regions. The intensity of the CS XRD peaks decrease for the CS/α-Ag 2 WO 4 composites (Fig. S2B), which was also observed by Khan et al. 49 . Moreover, the inset in Fig. S2B shows an increase in peak intensity at 2θ = 28.8° for all composites, indicating a preferential growth of the α-Ag 2 WO 4 structure towards the (3 0 1) crystallographic plane.
Fourier-transform infrared spectroscopy (FTIR) was carried out to evaluate the vibrational modes of the assynthesized CS/α-Ag 2 WO 4 composites. Figure S3 displays the characteristics bands of the CS polymer. Bands related to the (-C=O) carbonyl group (2000-1650 cm −1 ) cannot be observed, indicating no degradation of the CS polymer after fs laser irradiation 49 . For the CS/α-Ag 2 WO 4 composites, the CS polymer bands present lower intensity. In addition, bands ascribed to [WO 4 2− ] and [AgO x ] (x = 2, 4, 6, and 7) can be considered constituent clusters of the α-Ag 2 WO 4 structure (Fig. S4). Figure 1A-C show field emission scanning electron microscopy (FE-SEM) images of the CS/α-Ag 2 WO 4 composites, where it can be seen that the CS polymers with α-Ag 2 WO 4 microcrystals are dispersed under and over the film. Several α-Ag 2 WO 4 irregular rod-like structures with hexagonal face www.nature.com/scientificreports/ of several sizes can be observed for all samples 50,51 , besides a covering around each microcrystal formed by the CS polymer. In the CS film, it is possible to note a ribbing forming root-type structures and different morphologies, such as pointed spear-like structures, which differ in shape depending on the CS solution concentration. While for the CS3.3/α-Ag 2 WO 4 composite roughness layers can be observed on the surface (Fig. 1A), for the CS6.6/α-Ag 2 WO 4 composite the surface is, smoother (Fig. 1B). In contrast, for the CS9.9/α-Ag 2 WO 4 composite, different roots can be found forming a foliage-type structure (Fig. 1C). Energy-dispersive spectroscopy (EDS) analysis performed on the α-Ag 2 WO 4 rod-like structures confirmed the presence of the elements Ag, W and O. On the other hand, only Ag was observed in the ribbing, root and spear-like structures (Fig. S5). These structures were formed due to fs laser beam and the effect of CS polymer, which promoted the reduction of Ag + to Ag 0 (AgNPs) 15,30,[52][53][54][55][56][57][58] . Figure 2 shows the transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM) images of the CS/α-Ag 2 WO 4 composites. The particles formed on the surface of the CS polymer are composed of larger α-Ag 2 WO 4 NPs and smaller AgNPs, as proved by HR-TEM and EDS analyses. According to Murugadoss et al. 59 , the AgNPs are stabilized due to the excess of -NH 2 and -OH groups present in the CS polymer chain (Fig. S6A-C).

Biocidal analysis. Evaluation of the minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC).
Initially, no statistical difference between the data obtained from vehicles (CS and AA) and the control (CT) without treatment (data not shown) was observed, which means that the vehicles did not interfere with the viability of the microorganisms tested and that the activity observed was solely due to fs laser irradiation of the CS/α-Ag 2 WO 4 composites. The literature describes that pure CS (capping agent), as well as AA (used to dissolve CS), has inhibitory activity against different species of microorganisms 63,64 . However, it was verified that when used in low quantity these vehicles did not present antimicrobial activity.
In a previous study, our research group 15 showed that α-Ag 2 WO 4 irradiated by fs laser can increase its biocidal activity when compared to the α-Ag 2 WO 4 . In this work, it was observed that the antimicrobial activity was dependent on the CS concentration. The MIC was determined by visual inspection (Table 1). It was found that the CS3.3/α-Ag 2 WO 4 and CS6.6/α-Ag 2 WO 4 composites presented similar activity (MIC ranging from 0.49 to 1.95 µg/mL) for all microorganisms tested, while the CS9.9/α-Ag 2 WO 4 composite increased onefold against the microorganisms S. aureus and C. albicans (3.9 and 0.98 µg/mL, respectively). The MFC/MBC were determined for the activity capable of inhibiting 99.9%. Therefore, it was possible to observe a concentration increase in relation to the MIC necessary to reach more than 90% inhibition. This increase was represented as "fold change" in Table 1, which displays the MIC value, together with the MFC/MBC values and their respective inhibition index (%) according to CFU/mL normalized by the CT group.
As a consequence, all CS/α-Ag 2 WO 4 composites irradiated by fs laser were more effective against Gramnegative (E. coli) than Gram-positive (S. aureus) bacteria and the yeast (C. albicans). This difference can be explained by the structure of the cell wall, which in the Gram-positive bacteria and the yeast is composed of peptidoglycan and teichoic acid, bringing more stability to the cell wall. Studies suggest that silver ions are able to attach to the membrane surface of the microorganism, leading to membrane disruption and increasing its permeability. As a result, they could enter cells, condensing DNA and reacting with proteins. Moreover, thiol groups, which are responsible for enzyme activity, are inactivated by reacting with silver 64,65 .
Accordingly, a greater efficacy was also observed for the E. coli, a Gram-negative bacteriuma. Its membrane is composed of lipopolysaccharides (LPS) containing phosphate and pyrophosphate groups that make the cell surface negatively charged. Additionally, as CS is a cationic polymer it facilitates the binding of ions to the membrane, causing the microorganism inactivation, as previously described 55 . In another study with E. coli, researchers reported that silver ions trigger the separation of DNA strands and weaken the link between protein and DNA, thus altering vital processes for the microorganism 66 . Therefore, the CS6.6/α-Ag 2 WO 4 composite was considered to have the best antimicrobial activity, as it exhibited low concentrations for the microbicidal effect and still presented a little difference between the MIC and MFC/MBC values (low fold-change value).
Cytotoxicity analysis. In this section we evaluate the cytotoxicity of CS/α-Ag 2 WO 4 composites irradiated by fs laser with the aim of developing an agent with antimicrobial activity for biomedical application. According to the literature, CS solutions exhibit toxicity depending on the dose and synthesis method 67 . Jena et al. 68 observed that the cytotoxicity of CS with AgNPs (CS-AgNPs) is dose-dependent and that the cell viability decreases as the concentration increases. The same cell behavior was observed in this study with CS/α-Ag 2 WO 4 composites irradiated by fs laser. However, the authors did not find any decrease in cell viability when evaluating only CS 68 , which may be explained by the CS synthesis method.
Herein, the cytotoxicity profile of different concentrations of CS and AA vehicles was evaluated in the NOKsi lineage cell by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Alamar Blue Table 1. MIC (µg/mL) and MFC/MBC (µg/mL) of CS/α-Ag 2 WO 4 composites irradiated by fs laser in three different CS concentrations (g/L) against the microorganisms C. albicans, S. aureus and E. coli, and the inhibition index (%) calculated according to CFU/mL data and normalized by control without treatment (n = 8). www.nature.com/scientificreports/ assay. In the analysis by MTT assay (Fig. S7), it was observed that after 24 h of contact ( Fig. S7A In general, we observed that the AA vehicle did not present a cytotoxic profile and that the CS6.6 vehicle presented the minimal cytotoxic dilutions for the MTT assay. The cytotoxicity profile analysis by MTT assay of the CS/α-Ag 2 WO 4 composites was carried out at different concentrations (Fig. 3). The results revealed that after 24 h (Fig. 3A,D,G) all composites presented statistical difference in relation to CT, with exception of the CS3.3/α-Ag 2 WO 4 composite (Fig. 3A), which showed no statistical difference in relation to CT at the three last concentrations (1.95-0.49 µg/mL). However, after 72 h (Fig. 3C,F,I) the cytotoxicity profile of the composites experienced some changes. No statistical difference in relation to CT was observed in the lowest concentrations for CS6.6/α-Ag 2 WO 4 and CS9.9/α-Ag 2 WO 4 (7.8-0.49 µg/mL), as well as for CS3.3/α-Ag 2 WO 4 (3.9-0.49 µg/mL). We can then conclude that the CS6.6/α-Ag 2 WO 4 composite presented the best non-cytotoxic profile since the vehicle CS6.6 did not show cytotoxicity for the last 5 dilutions (C3-C7), corresponding to 7.81-0.49 µg/mL concentrations of the CS6.6/α-Ag 2 WO 4 composite, which also presented no cytotoxicity.
In contrast, when the cytotoxicity profile of the CS and AA vehicles was evaluated by Alamar Blue assay (Fig. S8), we did not observe any statistical difference in relation to CT, independent of the incubation time, which means that the vehicles did not present any cytotoxic profile.
When the cytotoxicity profile of the CS/α-Ag 2 WO 4 composites was analyzed at different concentrations by the Alamar Blue assay (Fig. 4), it was noted that after 24 h of contact only the 31.25 µg/mL concentration for the CS3.3/α-Ag 2 WO 4 and CS6.6/α-Ag 2 WO 4 composites presented statistical difference (  www.nature.com/scientificreports/ ( Fig. 4F). Therefore, we can infer that the CS6.6/α-Ag 2 WO 4 composite presented the best non-cytotoxic profile, as it maintained the profile of less cytotoxicity for longer time, considering that any dilution of the CS6.6 vehicle showed cytotoxicity. Although in both methods the reagents are metabolized by mitochondrial enzymes and present in the cytoplasm, it is known that the MTT reagent is more metabolized by mitochondrial enzymes than the Alamar blue assay, thus providing important information on the influence of the compound on the cell 69 . Through data analysis it was possible to observe that the Alamar blue assay showed high viability index higher concentrations. These indexes were stable during the three incubation times, demonstrating that the composites did not damage the pathway through which the Alamar blue reagent is metabolized 70 . In contrast, the MTT assay showed low viability index at lower concentrations in the first 24 h of treatment, whereas in the periods of 48 and 72 h it was possible to observe a small cell recovery, which increased the viability index at higher concentrations, reaching the concentrations observed in the Alamar blue assay after 72 h of incubation. This profile suggest that the composites initially induce a stress in the mitochondrial metabolism without causing any damage to cell, as evidenced by their recovery. Therefore, we believe that it is important to show the results of both methodologies, elucidating that despite the oxidative stress generated (which is already known by the microcrystal), this is not a determinant for the loss of cell viability.
SARS-CoV-2 inactivation by the CS6.6/α-Ag 2 WO 4 composite. The 4.0 µg/mL concentration of the CS6.6/α-Ag 2 WO 4 composite was selected for SARS-CoV-2 antiviral assays due to its greatest efficiency in the inhibition of bacterial and fungal growth, as well as its best non-cytotoxic profile.
The viral titer in the cell supernatants was quantified by PFU/mL assay at 1 and 24 hpi (hours post infection) to study the effect of the CS6.6/α-Ag 2 WO 4 composite against virus inactivation (Fig. 5). SARS-CoV-2 titer at 1-hpi supernatants was equivalent to 4.0 × 10 3 PFU/mL when the cells were incubated with the virus exposed to CS6.6 or PBS, used as controls. Initially, the CS6.6/α-Ag 2 WO 4 composite reduced the viral titer quantification to 0.8 × 10 3 PFU/mL, an inhibition of virus infection in 80% of the controls. After 24 h of exposure to the inactivated virus solution with the CS6.6/α-Ag 2 WO 4 composite, the viral titer quantified in the cell culture supernatant was 41% and 52% lower than that quantified in the cell culture supernatants from exposure to the inactivated virus solution with CS6.6 and PBS, respectively (Fig. 5A). This reduction in the viral titer may reflect the SARS-CoV-2 virucidal effect promoted by the CS6.6/α-Ag 2 WO 4 composite. Despite this reduction, the number of virus RNA copies recovered from the infected cells under different treatments were not changed either at 1 or 24 hpi (Fig. 5B). These results indicate that the exposure to the CS6.6/α-Ag 2 WO 4 composite inactivated viral infection www.nature.com/scientificreports/ even though the identification of the pathogen through its genes was enabled. CS products such as cationically modified derivatives can inhibit human coronavirus replication 71 , and this inhibitory effect was observed in viral titer quantification from cell culture supernatant exposed to CS6.6 24 hpi (Fig. 5A).

Morphological analysis by TEM of Vero-E6 cell cultures infected with SARS-CoV-2 untreated
and treated with CS6.6/α-Ag 2 WO 4 . Vero-E6 cells untreated and treated with CS6.6/α-Ag 2 WO 4 composite (controls). In ultrastructural analyses of untreated Vero-E6 cells and analyzed after 24 h of cultivation, no morphological alterations were observed (Fig. 6A-C). In cells analyzed 24 h after CS6.6/α-Ag 2 WO 4 treatment, several changes were observed in the cytoplasm, such as proliferation of vesicles, vacuoles, numerous structures with concentric membranes (myelin figures) and changes in mitochondria (Fig. 6D-F), which are indicative of cellular stress. The formation of syncytia (a large cell-like structure formed by joining two or more cells) was also noted (Fig. 7A,B). In cells infected with SARS-CoV-2 and treated with CS6.6, vacuoles and proliferation of vesicles were observed (Fig. 7C,D). A greater number of ultrastructural alterations was found in cells infected with SARS-CoV-2 and treated with the CS6.6/α-Ag 2 WO 4 composite. The alterations most commonly observed in this case were vacu-  (Fig. 8A), besides thickening of the rough endoplasmic reticulum (data not shown). In addition, SARS-CoV-2 particles attached to the plasmatic membrane and in the cytoplasmic vesicle lumen were also observed (Fig. 8B). Cells infected with SARS-CoV-2 and treated with CS6.6 presented in their cytoplasm vacuoles, vesicles and numerous myelin figures (Fig. 8C). Virus particles   Proposed mechanism for the biocidal activity. The proposed biocidal mechanism of the CS/α-Ag 2 WO 4 composites is summarized in Fig. 9. It is possible to note that CS presents strong affinity with metal ions as a result of the presence of -OH and -NH 2 groups, which can reduce Ag + ions to AgNPs 55 . Thus, due to the consequent interaction with the fs laser irradiation, the AgNPs are formed in the system. According to Jena et al. 68 , the presence of AgNPs lead to the formation of reactive oxygen species (ROS), causing DNA damage, and consequently producing changes in its conformation. As a result, the aforementioned composites absorb the incident photons, and the electrons (e − ) in the VB are excited to the CB; at the same time, holes (h•) are generated in the VB. Moreover, the presence of AgNPs increases the population of e − in the CB of the semiconductor due to their surface plasmon resonance (SPR) effect, causing an accumulation of positive vacancies in the VB. The strong SPR effect of AgNPs in these composite systems helps to effectively transfer the photogenerated carriers, thereby facilitating the charge separation at the composite interface, drastically improving the biocidal activity of the composite compared to that of the counterparts. Therefore, the enhanced presence of h• in the VB causes a strong interaction with the H 2 O molecule, leading to the formation of •OH and H + . Simultaneously, the O 2 molecule is converted into •O 2 − in the CB of the semiconductor due to the reaction with e − . In addition, the protonation of •O 2 − renders the •O 2 H radical. It is reported that the oxidative stress is caused by imbalances in the production and elimination of ROS, resulting in biocidal activity 18 . It also prevents the vital function of the cell, affecting the viability, proliferation and redox status of various cell types 41 , thus destabilizing cell wall and membrane, and consequently leading to, cell death 27 .

Conclusions and outlook
The rapidly spreading outbreak of COVID-19 has challenged the world's healthcare sector over the last year. Thus, it has become crucial to trap and eradicate SARS-CoV-2 by using new materials. In this work, we reported the synthesis of chitosan/α-Ag 2 WO 4 composites generated by femtosecond laser irradiation. This material is very efficient to eliminate bacteria (Escherichia coli, Methicillin-susceptible Staphylococcus aureus, and the yeast strain Candida albicans) and SARS-CoV-2 by contact. This study offers a general strategy to construct biocide materials. The biomimetic function of CS/α-Ag 2 WO 4 composites in defeating COVID-19 transmission is promising. However, further studies are still necessary for developing new technologies based on the functionalization of this composite applied on protective materials and communal objects (e.g., mask, door handles, elevator buttons, gas pumps, and handrails) to reduce both disease transmission and fear of touching objects.    85 , the batch configuration was selected due to the simplicity of the technique and the lack of a requisite for a pumping device. In the batch processing the dispersed CS/α-Ag 2 WO 4 composites was contained in a glass cell, the laser beam was focused perpendicular to the surface and during irradiation a magnetic stirrer was used to expedite the movement and prevent gravitational settling. The setup is shown in Fig. S1. To find the right parameters of irradiation, different parameters were tested with smaller sample volumes. Finally, a laser beam of 6 mm in diameter, 1/e 2 criteria, mean power of 150 mW and irradiation during 2 h were found to be the optimum parameters to complete the CS/α-Ag 2 WO 4 processing. www.nature.com/scientificreports/ sidered the lower concentration without visual growth. The MBC/MFC values were determined by cell recovery in an agar culture medium. For that, the MIC and 5 concentrations higher than the MIC were submitted to tenfold serial dilution in PBS. Each dilution was plated by microtip methodology on petri dishes with specific agar medium, and the plates were incubated at 37 °C, overnight. Counting the number of colonies was carried out in the lowest possible dilution. The data were converted to Log 10 (UFC/mL) and converted in inhibition index related to control without treatment. The literature describes MFC/MBC as the minimum concentration of the antimicrobial agent capable of killing 99.9% of the number of colonies (CFU/mL) or reducing 3 units of Log 10 in relation to the untreated control [87][88][89][90] . As control were used microorganisms in standard culture (CT), and the vehicles (CS and AA) at the same concentrations of experimental groups (to evaluate their interference in the cell viability). This experiment was performed in quadruplicate and in two different occasions (n = 8). MTT assay. This assay assesses the rate of viable cells by mitochondrial activity (vitality assay) by quantifying tetrazolium salt reduction to formazan crystals, which occurs mainly by succinic dehydrogenase enzymes in mitochondrial fraction. After the incubation time (24, 48 and 72 h), the supernatant was removed and it was added 100 µL/well of MTT solution (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; 1.2 mg/mL; Sigma-Aldrich) in RPMI-1640 medium without phenol red (Sigma-Aldrich). The plate was incubated at 37 °C, 5% CO 2 . After 4 h, the supernatant was discarded and 100 µL/well of isopropanol (Synth) were added to solubilize the formazan crystals. Each well was homogenized, and the plate was submitted to analysis in a spectrophotometer at 540 nm. This protocol was performed at 24, 48 and 72 h of incubation.

Cytotoxicity assays in NOK
Alamar blue assay. In this assay, the viability rate was quantified by the metabolic activity of viable cells. The reagent has the compound resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide/LifeTechnologies) which is a non-fluorescent blue dye. This is reduced by reductase enzymes, present in the cytosol and mitochondria, to a highly fluorescent pink dye, resorufin. After 20 h of the cells being challenged with the CS/α-Ag 2 WO 4 composites, 20 µL of the alamar blue solution was added to each well. The plate was incubated for 4 h at 37 °C, 5% CO 2 and the analysis in a spectrophotometer at 540 nm (600 nm reference filter) for 24 h was performed. The same plate was incubated at 37 °C, 5% CO 2 to perform the analysis of times 48 and 72 h, since resazurin is a noninvasive and stable probe.
Statistical analysis for cytotoxicity assay. Shapiro-Wilk and Levene's test were performed to test data distribution and homogeneity. Based on normal and heteroskedastic distribution, statistical comparisons were performed by one-way analysis of variance (ANOVA) with Welch correction, followed by Games Howell Post Hoc, using BM SPSS Statistics program (version 23). All data are plotted as the mean + standard deviation (SD) and p < 0.05 was considered statistically significant. www.nature.com/scientificreports/ 1 and 24 hpi. For this purpose, Vero-E6 cells (1.5 × 10 6 ) previously seeded, maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) in cell culture 24 cm 2 flasks, were incubated with each viral inactivated solution in multiplicity of infection (MOI) 0.01 for 1 h at 37 °C and 5% CO 2 . Then, the supernatants were harvested and the cell monolayers washed twice with PBS and physically removed from the flasks for processing for transmission electron microscopy (TEM) analysis, or incubated with DMEM/HEPES/2% FBS for 24 h, before harvesting the supernatant and preparation to TEM. The supernatants were stored at − 70 °C for posterior virus titration and RNA quantification by Plaque Forming Units (PFU/mL) and qRT-PCR (number of copies/mL), respectively 93,94 .
Virus titration and RNA quantification. For PFU assay, monolayers of Vero-E6 cells (10 5 cells/well) were seeded into 24-well culture plates (flat bottom) and grown for 24 h at 37 °C in 5% CO 2 . These cells were inoculated with 300 µL of infected cells supernatants dilutions (10 −1 to 10 −4 ). After 1 h at 37 °C in 5% CO 2 , the medium was changed to 500 µL of a solution containing DMEM-High glucose 1X, 1.8% carboxymethylcellulose and 2% FBS. 72 h post infection, cytopathic effects (CPE) were observed on optical microscope and cells fixed with 10% formalin. After 3 h, this solution was removed and monolayers stained with 0.04% crystal violet for Plaque Forming Units counting 93 . The molecular detection of viral RNA levels was performed as described before 94 . Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect the SARS-CoV-2 envelope gene (E) 95 . Cell supernatants were used for viral RNA quantification by real time qRT-PCR and were expressed in number of copies of virus RNA per mL.
Concurrently to viral RNA amplification, standard curves were plotted with different numbers of copies per cycle threshold (Ct). The standard curve method was used in comparison with the viral gene to obtain the relative quantification of the viral RNA in supernatants 96 .

Transmission electron microscopy (TEM).
For TEM analyses the Vero-E6 cells suspensions were fixed in 2.5% glutaraldehyde in sodium cacodilate buffer (0.2 M, pH 7.2), post-fixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin and polymerised at 60 °C over the course of three days 81,82 . Ultrathin sections (50-70 nm) were obtained from the resin blocks. The sections were picked up using copper grids, stained with uranyl acetate and lead citrate 83 , and observed using Hitachi HT 7800 transmission electron microscope.
Statistical analysis for antiviral assays. The data were analyzed using Past program 97 . The one-way ANOVA with Tukey post-test was performed to determine the significance between the different experimental groups (CS6.6/α-Ag2WO4, CS6.6 and PBS). The graphics were plotted by the OriginLab Pro 2021 program.
Results are presented as the mean of 3 independent experiments ± standard deviation (SD) with a confidence interval of 95%, and significant p values were represented as d for < 0.005 and e for < 0.05.

Supporting Information
Detailed information about the XRD, FTIR, FE-SEM, TEM, and cytotoxicity discussion are described in the Electronic Supporting Information. www.nature.com/scientificreports/