key: cord-0781223-zilh7z58
authors: Brault, Ariane; Néré, Raphael; Prados, Jérôme; Boudreault, Simon; Bisaillon, Martin; Marchand, Patrick; Couture, Patrick; Labbé, Simon
title: Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant protect Vero E6 cells against infection by viral pseudotyped particles expressing SARS-CoV-2, SARS-CoV or MERS-CoV Spike protein
date: 2022-03-23
journal: bioRxiv
DOI: 10.1101/2022.03.22.485373
sha: 0f7ad35a3d46ae6b263c0ef46b695e217cf41802
doc_id: 781223
cord_uid: zilh7z58

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses (HuCoV) that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surface and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S- pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:16 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

The emergence of the novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a severe public health burden worldwide. [1] [2] [3] [4] The fast spread of SARS-CoV-2 pandemic poses a striking challenge for public services and health facilities. Public spaces and healthcare environments become rapidly contaminated by way of person-to-person contact.

Several studies have shown that the main way by which people are infected with SARS-CoV-2 is through respiratory droplet transmission and airborne spread. [5] [6] [7] [8] [9] Individuals can also be infected with SARS-CoV-2 through contact with surfaces and inanimate materials, called fomites.

However, the risk of surface-mediated transmission is dependent on several factors. [10] [11] [12] [13] [14] [15] Among these are the infection prevalence rate in the community, the density of virus particles present on the surfaces, the time between viral contamination of the surface and transmission to the people, and the amount of virus particles from surfaces to hands and from hands to the mucous membranes of the nose, mouth, and eyes.

Coronaviruses including SARS-CoV-2 remain viable on a variety of porous and non-porous surfaces. 10, [16] [17] [18] Although these viruses are rapidly eliminated on porous surfaces within minutes to few hours, they are more persistent on non-porous surfaces such as plastic, glass, and stainless steel in which case they can remain infectious up to 3 days. 10, 17, 19, 20 In contrast, SARS-CoV-2 is rapidly inactivated after 4 h on copper (Cu) surfaces. 18 Interestingly, yeasts, bacteria, and viruses are also rapidly killed on surfaces of Cu or Cu alloys containing at least 70% Cu. [21] [22] [23] This process is called Cu-mediated "contact killing". 21 Importantly, it has also been known that concentrations of Cu required to kill microbes are not toxic to humans. 24 The insensitivity of human tissue to Cu can be contrasted with microorganisms that are extremely sensitive to its toxic effects. 25 One exciting new application of nanotechnology consists of using carboxymethyl cellulose (CMC) as a nanosubstrate to generate Cu-containing nanoparticles using copper sulfate and sodium borohydride. 26, 27 The resulting cellulosic cuprous nanoparticles (CMC-Cu) possess antimicrobial activity against non-pathogenic microbes such as the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. 26, 28, 29 Biophysical analysis of CMC-Cu nanoparticles have revealed that Cu is incorporated into the CMC polymer in its reduced state (Cu 1+ ). 27 Electron microscopic analyses have shown the presence of spherical particles on the surface of the CMC film. 26 Cu is thought to be slowly released from these particles under its reduced state (Cu 1+ ) and becomes highly toxic as it changes its oxidative state. This redox active process is predicted to be associated with generation of destructive hydroxyl radical and superoxide anions. These reactive oxygen species (ROS) have the potential to cause detrimental oxidative damage to vital microbial cell constituents such as lipids (including those in the cell membrane), proteins and nucleic acids. For example, in the case of S. cerevisiae, results have shown that it is more sensitive to CMC-Cu nanoparticles than soluble CuSO4. 28 Yeast cells treated with CMC-Cu are more susceptible to lipid peroxidation than untreated cells. Furthermore, the CMC-Cu-treated cells exhibit morphological anomalies that are indicative of cell surface damage and loss of membrane integrity. 29 These studies suggest the potency of CMC-Cu as sanitizing agent to prevent contamination of fomites by enveloped coronaviruses is of interest.

An efficient way to prevent indirect community transmission of coronaviruses by contaminated surfaces consists of using disinfectants and cleaning agents against enveloped viruses. 30 World Health Organization (WHO) recommends that disinfectants must contain 70% ethanol or isopropanol, or 0.5% hydrogen peroxide and, must be used at least, a 20 s treatment. 31, 32 One caveat of using alcohol-based disinfectants is their high volatility. 33 To favor the time of retention of alcohol-based disinfectants on inanimate surfaces, anionic surfactants such as sodium dodecylbenzenesulfonate and sodium laureth sulfate must be added. 33 Moreover, owing to their intrinsic nature that includes both hydrophobic and hydrophilic properties, anionic surfactants possess the advantage that they can dissolve the outer lipid layer of enveloped viruses, while remaining soluble in water as well as in alcohol-and H2O2-based disinfectants. 34, 35 Studies using native coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV are restricted to biosafety level 3 (BSL-3) containment facilities. 36 However, alternative models have been developed that allow production of replication-deficient enveloped virus-like particles that are safer surrogates of native coronaviruses. [36] [37] [38] [39] One useful and safe model is to use murine leukemia virus (MLV) core components to form the enveloped pseudovirion backbone. 36, 38 During its formation, for instance in HEK-293T cells, a second expression plasmid triggers the synthesis of a luciferase reporter transcript flanked by retroviral regulatory LTR regions and a packaging signal that allow incorporation of the reporter mRNA into a nascent virus-like particle.

Moreover, during the assembly process a third plasmid encodes either SARS-CoV-2, SARS-CoV, In this report, SARS-CoV-2-, SARS-CoV-and MERS-CoV-S-pseudotyped particles were rapidly inactivated upon exposure to CMC-Cu. That resulted in a dramatic decrease of their potency to infect target cells. Similarly, infectivity of these three types of S-pseudotyped particles was dramatically reduced when they had been pretreated with the disinfectant Saber TM over a 30 s time period. Of significance, results showed that a pretreatment with Saber TM in combination with CMC-Cu was the most effective strategy to prevent infectivity of SARS-CoV-2-, SARS-CoV-and MERS-CoV-S pseudovirions as well as pseudoviral particles expressing a relevant variant form of SARS-CoV-2 S protein.

Cell culture. HEK-293T (human) and Vero E6 (African green monkey) cells were grown in Dulbecco's modified Eagle's medium (DMEM; Wisent). DMEM medium was supplemented with 10% fetal bovine serum (FBS; Wisent) and 1X penicillin-streptomycin (PS) solution (Mediatech).

Cell lines were grown at 37 o C in the presence of 5% CO2 under a humidified atmosphere.

Transfection of HEK-293T cells was carried out using Lipofectamine 2000 (Thermo Fisher Scientific -Invitrogen). Transfection conditions used a ratio of 1 µg total DNA (including all three plasmids) for 3 µl Lipofectamine 2000 that were mixed in the presence of Opti-MEM (Life Technologies) as described previously. 36, 38 Cell viability was determined by a colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described previously. 40 SARS-CoV-2-S mutant alleles were created to substitute the codons corresponding to Asn501 and Asp614 with Tyr and Gly codons, respectively. These N501Y and D614G mutants were generated by a PCR overlap extension method. 42 DNA sequences containing site-specific mutations from each respective PCR-amplified fragment was digested with NotI and ApaI and exchanged with a corresponding DNA region into the pcDNA3.1-CD5-SARS-CoV-2-S-Δ19.

Synthesis of Luciferase (LUC) RNA molecules for production of standard curves. Using plasmid pTG-Luc as a template, the DNA sequence encoding LUC was isolated by PCR with primers containing KpnI and BamHI sites. 36 The PCR product was cloned into the corresponding sites of pBluescript SK in which a T7 promoter sequence is found immediately next to the cloning site on the 5' end. The resulting plasmid was named pSK-Luc. Using BamHI-linearized pSK-Luc (5 µg per reaction), in vitro run-off transcription with T7 RNA polymerase was performed using buffer T (80 mM HEPES, pH 7.5, 20 mM MgCl2, 2 mM spermidine, 40 mM DTT and 10 mM NaCl) that was supplemented with pyrophosphatase (0.1 U/µl) as described previously. 43 The DNA template was eliminated by digestion with RNase-free DNase I for 15 min at 37 o C. LUC RNA molecules were purified using preparative polyacrylamide gel electrophoresis as described previously 44, 45 . Subsequently, RNA molecules were extracted from the gel using the crush and soak method. 45 Production of S-pseudoviral particles. The biological system to produce S-pseudotyped viral particles was based on a replication-deficient murine leukemia virus (MLV) that lacked essential genes to achieve a complete replication cycle. 36, 38 Furthermore, the genome of viral particles was modified to encode the LUC reporter gene, thereby allowing measurement of infectivity of the S-pseudoviral particles after host cell infection. For production of MLV-based pseudoviral particles, HEK-293T cells were co-transfected with three plasmids as described previously. 36 harboring the desired S protein as described previously. 36, 38 Cells were incubated on a rocker for 2 h and then supplemented with complete DMEM (300 µl) medium and incubated for 72 h.

Infected cells were lysed as described previously. 36, 38 Lysed cell supernatant from each well was assessed for luciferase activity using the Luciferase Assay System (Promega). Relative luciferase units (RLU) were determined using the Glomax 20/20 luminometer (Promega) and infectivity values were analyzed as described previously 36 and plotted using GraphPad Prism 9.3.1 software.

carboxymethyl cellulose (CMC) (0.2 g) was dissolved in 9.8 ml of sterile MilliQ water under vigorous magnetic stirring conditions as described previously. 26 

During the production of MLV-based virus-like particles, we assumed that the RNA molecule being encapsulated into pseudoviral particles contained only one copy of the coding sequence of the luciferase (LUC) gene reporter. Based on this assumption, we isolated the coding region of LUC and subcloned it in a way that a T7 promoter sequence was located at its 5' end. The resulting DNA template was used to generate synthetic LUC transcripts that were utilized as standards in RT-qPCR analysis to produce standard curves by linear regression (Fig. 1A ). This approach allowed us to evaluate the number of LUC RNA transcripts that were present in any given batch of pseudoviral particles. This in turn allowed calibration and showed that VSV-G and S transcripts were expressed at both times ( Fig. 2A) . Although S transcript levels exhibited a slight reduction at 48 h compared to 24 h, their mRNA levels remained robustly expressed. In the case of VSV-G transcripts that were detected at 48 h, results showed that their levels were reduced 3.7-fold less than those observed at 24 h ( Fig. 2A) . However, this decrease was not sufficient to prevent detection at 48 h of both VSV-G mRNA and protein steady-state levels (Fig. 2B ).

S-pseudoviral particles were retrieved after 48 h of transfection to further validate the presence of S proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV. Purified virus-like particles were analyzed by immunoblot assays. Results showed that S proteins were present in SARS-CoV-2, SARS-CoV, and MERS-CoV pseudovirions (Fig. 2B ). In the case of SARS-CoV-2-S and MERS-CoV-S, the uncleaved (S0) form and the band corresponding to the S2 segment (S2) were detected as previously reported (Fig. 2B) . 48, 49 The presence of the lower molecular weight band S2 could have resulted from the action of furin or some related proprotein convertase(s) activity during the process of maturation that occurs within the secretory pathway in HEK-293T cells. Weakly detected higher order bands may correspond to oligomeric forms of S protein. 48, 49 VSV-G and S proteins were undetectable in Δenv pseudoviral particles that are devoid of viral envelope glycoproteins (Fig. 2B) . Production of the p30 capsid protein of MLV was co-currently followed as a control for each pseudotyped particle preparation. This approach was used to validate the presence of an additional viral protein produced and incorporated into MLV-based pseudoviral particles. Taken together, these results indicated that pseudoviral particles contain the viral envelope VSV-G or S protein that is required to infect Vero E6 cells. h of incubation, cell viability was assessed by a MTT method (Fig. S1, A) . 41 The calculated concentration of Saber TM that resulted in half maximal inhibitory concentration (IC50) was 1.48%, whereas 1.0% and lower concentration of Saber TM did not significantly affect Vero E6 cell viability Similarly, in the case of SARS-CoV-S pseudotyped particles, a pretreatment with 0.4% or 1%

Saber TM for 30 s or 15 min strongly inhibited their ability to infect Vero E6 cells (Fig. 3E) . Results showed that cell extracts from Vero E6 cells that had been exposed to Saber TM -treated SARS-CoV-S pseudoviral particles exhibited 98.7% to 99.7% less luciferase activity than conditions without Saber TM treatment (Fig. 3E) . MERS-CoV-S pseudoviral particles also exhibited hypersensitivity to 0.4% and 1% concentrations of Saber TM treatment for 30 s or 15 min (Fig. 3F) .

Utilization of Saber TM -treated MERS-CoV-S pseudoviral particles dramatically lowered the relative luciferase activity that was measured in cell extracts prepared from infected Vero E6 cells.

Saber TM concentration as low as 0.4% inhibited luciferase activity in target Vero E6 cells by 98.7%

( Fig. 3F) , whereas luciferase activity was reduced by 99.6% in the presence of 1% Saber TM (Fig.   3F ). Taken together, these results indicated that Saber TM is an efficient disinfectant to reduce SARS-CoV-2-, SARS-CoV-, and MERS-CoV-S-pseudotyped particle infection in a MLV-based pseudoviral system. To test whether treatment with CMC-Cu affected viability of Vero E6 cells, the cells were exposed Results showed that cells that had been exposed to CMC-Cu-treated SARS-CoV-2-S pseudoviral particles (CMC-Cu at a concentration of 1:100) displayed 97.1% to 97.3% less luciferase activity compared to the untreated pseudoviral particles (Fig. 4D) . Furthermore, we observed a decrease of 99.6% to 99.9% less luciferase activity when Vero E6 cells were infected with pseudoviral particles expressing SARS-CoV-2 S protein that had been pretreated with CMC-Cu at a dilution of 1:50 and 1:25, respectively (Fig. 4D ).

In the case of SARS-CoV-S pseudovirions, a pretreatment with CMC-Cu (1:100) resulted in a strong reduction in their ability to infect Vero E6 cells as shown by a 86.1% to 90.3% reduced luciferase activity (Fig. 4E) . Therefore, we only used the SARS-CoV-2-S-pseudotyped virus for the next series of experiments.

The redox active nature of Cu makes it highly susceptible to engage in chemical reactions that generate hydroxyl radical, a free radical species that damages membrane lipids, proteins and nucleic acids. These observations led us to test whether the Cu chelator tetrathiomolybdate (TTM) would protect S-pseudovirions against the CMC-Cu nanotoxicity. As a control, results showed that incubation of S-pseudoviral particles with TTM (4 mM) alone did not affect their ability to infect Vero E6 cells. Indeed, lysates from these cells yielded high levels of luciferase activity that were set to 100% compared with low background levels of luciferase activity (0.2%) in cell extracts from cells infected with Δenv pseudoviral particles (negative control) (Fig. 6A) . When SARS-CoV-2-S pseudovirions were treated with CMC-Cu (1:100) over a period of time of 30 s, there was a strong inhibitory effect on their infectivity. Luciferase assays showed a 97.6%

reduction of activity compared to the absence of treatment (Fig. 6A) . In contrast, when S-pseudovirions were treated with CMC-Cu (1:100) and TTM (4 mM), levels of luciferase activity were restored to 95.6% compared to levels of S-pseudoviral particles not exposed to CMC-Cu ( Fig. 6A) .

To further investigate the mechanism of the action of CMC-Cu, we investigated the protective effect of Tiron, a hydroxyl radical quencher, to interfere with the inhibitory action of CMC-Cu nanoparticles. As described above, a treatment with CMC-Cu (1:100) within 30 s strongly inhibited the ability of S-pseudovirions to infect Vero E6 cells, resulting in luciferase activity that was 98.2% less than activity of untreated S-pseudovirions (CMC-Cu free) (Fig. 6B) . When Tiron and CMC-Cu were used together to treat pseudovirions expressing the SARS-CoV-2-S protein, the luciferase signal observed in Vero E6 cells was fully restored to 100% of that of reference Spseudovirions (Fig. 6B) . Taken together, these results suggested that the redox active nature of Cu was required to inactivate S-pseudovirions.

To further investigate the relationship between CMC-Cu and S-pseudoviral particles, Western blot assays were performed to examine the S protein levels of purified pseudovirions following their treatment with CMC-Cu after 30 s and 15 min. Results showed that the levels of S0 form of Spike were rapidly reduced when pseudovirions had been incubated with 4% CMC-Cu (dilution 1:25) (Fig. 6C) . Moreover, additional low molecular weight bands were present and increased in intensity as a function of time of treatment (Fig. 6C ). In the case of the S2 form, protein fragment levels appeared relatively stable in the presence of CMC-Cu. The capsid MLV p30 protein levels were probed as loading control. It is tempting to suggest that the rapid loss of S0 form integrity of the S protein may contribute to the dramatic reduction of infectivity of viral pseudotyped particles exposed to CMC-Cu. respectively, compared to levels of pseudoviral particles that expressed wild-type S protein (8.9 x 10 4 RLU) (Fig.7A) . However, their average values of luciferase activity in these instances were lower than in the case of cells infected with pseudoviral particles expressing the S 501Y/614G protein (Fig. 7A) .

During pseudotyped particle production in HEK-293T cells, we validated that S mutant proteins of SARS-CoV-2 were expressed at both transcriptional and posttranscriptional levels. We focused our attention on the S 501Y/614G mutant since pseudoviral particles expressing this variant form of S protein gave the highest luciferase signal. As shown in Fig. 7B , S 501Y/614G RNA transcripts were strongly detected in total RNA preparations from HEK-293T after 24 and 48 h of transfection. When pseudotyped particles were collected at 48 h time point, results showed that the S 501Y/614G mutant protein was consistently detected from pseudovirions (Fig. 7C) . The two forms of S 501Y/614G that were detected included the uncleaved (S0) form and the S2 segment (S2) of the protein as reported previously (Fig. 7C) . 48, 49 To assess the efficiency of Saber TM and CMC-Cu against S 501Y/614G pseudoviral particles, we incubated aliquots of these pseudovirions with Saber TM and CMC-Cu concentrations ranging from 0.0004 to 1% and 0.0005 to 4%, respectively. After 30 s, Saber TM -and CMC-Cu-treated S 501Y/614G pseudotyped particles were used to test Vero E6 cells infection by luciferase assays at 72 h (Fig. 8, A -B) . Results showed that half maximal inhibitory concentration (IC50) of Saber TM and CMC-Cu to block the infectivity of S 501Y/614G pseudotyped particles was 0.02% and 0.06%, respectively (Fig. 8, A -B) . These results indicated that viral pseudotyped particles were 74-and 170-fold more sensitive to Saber TM and CMC-Cu, respectively, than Vero E6 cells. (Fig. 8C) . background values with the lowest signal intensity (99.9% reduction) (Fig. 8E ). In contrast, control experiments revealed maximal luciferase activity in cell extracts prepared from Vero E6 cells infected with untreated S 501Y/614G pseudotyped particles. Taken together, these results revealed that Saber TM in combination with CMC-Cu remain highly effective to inhibit SARS-CoV-2 S-pseudotyped particle variants harboring 501Y, 614G, and 501Y/614G mutations.

Although SARS-CoV-2 infections mostly occur as a result of person-to-person contact through exposure to respiratory droplets, viral transmission can also occur from high-touch contaminated surfaces to hands, and from hand-to-face contacts as observed for other pathogens. 57 diameter of 80 to 150 nm. 26, 27, [61] [62] [63] In this way, the antimicrobial potency of Cu could be increased by the delivery of Cu in the form of CMC-Cu, therefore creating a "copperized nanosurface" that favors direct pseudovirus-Cu contacts, rendering Cu more effective at inactivating pseudovirions.

In support of this hypothesis, a previous work has shown that yeast cells exposed to CMC-Cu containing the equivalent of 157 µM Cu exhibit the same level of growth inhibition than the same cells exposed to soluble Cu in which case 400 µM of cupric sulfate is present, revealing that Cu exhibits a greater toxicity under the form of CMC-Cu than soluble Cu. 28 In addition, a report has shown that carboxymethyl cellulose (CMC) is the best support for catalyst Cu as it incorporates the highest amount of Cu per gram of nanomaterial due to its higher number of sodium-carboxyl groups. 64 Furthermore, analyses have shown that the oxidative state of Cu in the final nanomaterial product is Cu 1+ , making it able to engage in chemical reactions that generate hydroxyl radical, a free radical species that is cytotoxic.

In the case of pseudoviral particles used in our study, they consisted of a surrogate viral core with a heterologous coronavirus S protein at their surface. Previous studies have shown that these pseudoviral particles behave like their native coronavirus counterparts for entry steps into susceptible cells. 36, 38 Thus, use of these S-expressing pseudoviral particles represents an excellent when SARS-CoV-2-S pseudovirions were exposed to 0.4% and 1% of Saber TM for 30 s, ≥2.2-and 2.5-log10 inactivation was observed (Fig. S3) . These drops in reporter activity revealed that the most of the infectivity of S-pseudoviral particles was eliminated by 0.4% and 1% of Saber TM exposure of 30 s. These results were reminiscent of the effective inactivation of human coronaviruses (HCoV) by disinfection procedures with 70% EtOH, 70% isopropanol, 0.5% H2O2, or 0.1% sodium hypochlorite within a 30 s or 60 s exposure. 30, 67 Based on a previous study that reported enhanced virucidal effect of Cu by the addition of hydrogen peroxide 68 , we have investigated whether a treatment with CMC-Cu in combination with peroxide-based Saber TM potentiated the level of inactivation of S-pseudoviral particles.

Results showed that S-pseudovirions were more sensitive to the Saber TM -CMC-Cu mixture than to Saber TM or CMC-Cu alone. In the case of SARS-CoV-2 S-pseudotyped particles, a treatment with the Saber TM -CMC-Cu mixture resulted in a 2.8-log10 decrease which represented 100% reduction compared to background levels of infectivity observed with Δenv pseudoviral particles in Vero E6 cells assays (Fig. S4) .

A previous study has reported that 10-or 30-min exposure to a Cu brass surface resulted in morphological alterations to the human coronavirus 229E, including envelope damages and loss of the surface S protein. 23 

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We are grateful to Dr. Gilles Dupuis for critical reading of the manuscript and for his valuable