key: cord-350948-oog6m4h3
authors: Leclercq, Loïc; Nardello-Rataj, Véronique
title: How to improve the chemical disinfection of contaminated surfaces by viruses, bacteria and fungus?
date: 2020-09-17
journal: Eur J Pharm Sci
DOI: 10.1016/j.ejps.2020.105559
sha: 
doc_id: 350948
cord_uid: oog6m4h3

In response to the current pandemic situation, we present the development of an effective virucidal and biocidal solution to prevent from the spread of infectious diseases through contact with contaminated surfaces. The disinfectants, based on equimolar mixtures of didecyldimethylammonium chloride ([DiC(10)][Cl]), dodecyloctaglycol (C(12)E(8)), and cyclodextrin (CD), show synergistic effects against enveloped viruses (RSV, HSV-1, VACV) and fungi (C. albicans), and additive responses against bacteria (P. aeruginosa). These synergistic mixtures could then be highly helpful for prevention of respiratory illnesses, since a boosted activity allows: (i) a faster eradication of pathogens, (ii) a shorter contact time, and (iii) a complete and broad-spectrum eradication to avoid spread of resistant strains (including bacteria and fungi).

and cleaning of surfaces must be systematically implemented as a prevention measure of CoViD-19 (Dexter et al., 2020) . The disinfecting process requires the use of chemical biocides and virucides to inactivate pathogens on surfaces whereas the cleaning step is only used to remove pathogens, dirt and impurities. Both processes lower the number and the risk of transmission.

In this context, the didecyldimethylammonium chloride ([DiC 10 ] [Cl] ), one of the most widely used quaternary ammonium compounds in healthcare systems to prevent and control viral infections due to its ability to disorganize and to disrupt the envelopes of viruses (Leclercq et al., 2010) , is associated with detergents, such as dodecyloctaglycol (C 12 E 8 ), making the detergent-disinfectant combination ideal for general sanitation purposes (Rauwel et al., 2012) . As C 12 E 8 is able to solubilize the viral envelope via micellization, the C 12 E 8 potentiates the virucidal activity of [DiC 10 ] [Cl] due to the formation of synergistic co-micelles (Nardello-Rataj and Leclercq, 2016) . However, this solution only works with the enveloped viruses since they are more sensitive to destruction than bacteria, fungi and viruses without lipoprotein envelopes (Prince and Prince, 2001) . On the other hand, cyclodextrins (CDs) can be used as virucidal agents due to their interactions with the viral-lipids (Carrouel, 2020) . For example, methyl--CD alone reduces the infectivity of CoV (Pratelli and Colao, 2015) whereas native -CD boosts the virucidal action of [DiC 10 ][Cl] against enveloped viruses since both of them act on the viral-lipids .

It may be hypothesized that [DiC 10 ][Cl]/C 12 E 8 /CD systems would demonstrate synergistic effects against enveloped viruses as these compounds act on the same target ( Figure 1 ). In order to safe test the virucidal activities, respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), vaccinia virus (VACV) and coxsackievirus B4 (CVB4), were used instead of hazardous pathogens, including SARS-CoV-2. These synergistic mixtures could then be highly helpful for prevention of viral respiratory illnesses, since a boosted activity allows: (i) a faster eradication of viruses, (ii) a shorter contact time, and (iii) a complete and broad-spectrum eradication to avoid spread of resistant pathogens (e.g. bacteria and fungi).

Insert Figure 1 .

Didecyldimethylammonium chloride ([DiC 10 ] [Cl] ) was as synthesized according to the procedure described in our previous work (Leclercq et al., 2010) . Dodecyloctaglycol (C 12 E 8 ) was purchased from TCI (purity higher than 99.9%). The other chemical compounds were purchased from Sigma-Aldrich Chemical at the highest purity available. The compounds used in the biological assays, strains and cells were purchased as mentioned below. Sterile water was used in all experiments. Each solution was prepared extemporaneously. The composition of the neutralizer and diluent was: phosphatidylcholine, 3 g; Tween 80, 30 mL; sodium thiosulfate (5 H 2 O), 5 g; histidine chlorhydrate, 1 g; saponin, 30 g; tryptone salt, 9.5 g; water qs. 1 L. Experiments were assayed in triplicate.

HSV-1 (Strain Kos) and VACV (Strain Elstree) were propagated in Vero cells (ATCC® CCL-81™) in Minimum essential Medium (Gibco, Life Technologies) supplemented with 2 mM (l)-glutamine (Gibco), 1% non-essential amino-acids (Gibco) and 2% inactivated fetal calf serum (Gibco). The non-enveloped CVB4 (strain JVB) were propagated in BGM cells in the same medium. RSV (local laboratory strain) was propagated in Hep-2 cells (ATCC® CCL-23™) in the same medium. Cell-free viral suspensions of the viruses were obtained by freezing-thawing cycles followed by a low speed centrifugation to remove cell debris. Viruses titers were assayed by the cytopathic effect of serial dilutions (1:10) of virus-containing samples on Vero or Hep-2 cells. A sample (100 µL) for each dilution was used to infect four replicate wells in 96-well microtiter plates (NunclonTM Delta Surface, Thermo ScientificTM NuncTM). Virus-induced cytopathic effects were scored after 5 days of incubation at 37 °C ± 0.1 °C in a humidified 5% CO 2 atmosphere. Titers were expressed as the quantity of viruses infecting 50% of the tissue culture wells (Tissue Culture Infectious Doses, TCID 50 , Spearman, 1908; Kärber, 1931) . The detection limit was 5.62 TCID 50 /mL. Virus stocks were 1.3×10 7 TCID 50 /mL for RSV, 1.5×10 7 TCID 50 /mL for HSV-1, 1.1×10 7 TCID 50 /mL for VACV, and 6×10 6 TCID 50 /mL for CVB4. separate viruses from the other components of the mixtures. Residual viruses were then titrated as described above. Each experiment was performed at least twice. The virucidal activity was determined by the difference of the logarithmic titer of the virus control minus the logarithmic titer of the test virus. This difference is presented as a reduction factor including its 95% confidence interval. A reduction in virus titer of ≥4-log 10 was regarded as evidence of sufficient virucidal activity according to EN 14476. The lowest concentration giving a reduction in virus titer of ≥4-log 10 was the minimum virucidal concentration (MVC).

Various solutions of [DiC 10 ][Cl], C 12 E 8 and CD alone or in mixture were prepared and the critical micelle concentrations (CMCs) were estimated by surface tension measurements using the Wilhelmy plate method (Tensiometer K100, Krüss) at 25 ± 0.05 °C. All equilibrium surface tension values were mean quantities of at least three measurements. The precision of the force transducer of the surface tension apparatus was 0.1 mN m -1 and before each measurement, the platinum plate was cleaned in red/orange color flame. Variations of air/water surface tension as a function of total surfactant concentration were plotted. As the surface tension is linearly correlated to the surfactant concentration in logarithmic scale in both pre-and post-micellization regions, the intersection point of these two straight lines corresponds to the CMC (± 10% of the value).

The solutions were placed in light-scattering cells (10 mm) to determine the hydrodynamic diameter (D h ) using 3D LS Spectrometer (LS instruments) at 25 ± 0.05 °C. 3D cross correlation mode was used with two APD to improve the detection of small size micelle (< 5 nm) at 90°. Cumulant method was applied as data treatment of the correlogram and polydispersity index was in all case lower than 0.2. The analysis provides an average diffusion coefficient used to calculate a D h ± 0.2 nm using the Stokes-Einstein equation.

The measurements were performed using a Zetasizer Nano ZS (Malvern Instruments) at 25 ± 0.05 °C. Three runs per sample were used to establish measurement repeatability. The applied voltage can be set to automatic mode (start at a low voltage, typically 80 V, and increase the voltage gradually to 150 V. The electrophoretic mobility was determined experimentally and used to calculate the -potential.

The measurements were taken with a CDM210 conductivity meter (Radiometer). All measurements were taken at 25 ± 0.05 °C. As the conductivity is linearly correlated to the surfactant concentration in both pre-and post-aggregation regions, the ratio between the two slopes gives the apparent degree of micelle ionization ( ± 3 %).

All bactericidal tests were performed using Pseudomonas aeruginosa (ATCC® 15442™).

They were carried out in accordance with European standard EN 1040. A sample of the product was diluted with water and a test suspension of bacteria cells was added. The number of bacteria cells in the suspension was adjusted between 1.5×10 7 and 5.0×10 7 CFU/mL. The mixture was maintained at 20 ± 1 °C for 5 min ± 10 s. At the end of this contact time, an aliquot was taken; the bactericidal activity in this portion was immediately neutralized. The number of surviving yeasts in each sample was determined. Samples were serially diluted from 10 -1 to 10 -5 and each dilution was plated in duplicate on tryptone soya agar. After 48 h incubation of the plates at 37 °C, the colony-forming units par millimeter (CFU/mL) were counted. The bactericidal activity was determined by the difference between the logarithmic number of CFU/mL in the suspension at the beginning of the contact time (control test) and the logarithmic number of survivors per mL. This difference is presented as a reduction factor including its 95% confidence interval. A reduction of ≥4-log 10 (corresponding to a destruction of ≥99.99%) was regarded as evidence of sufficient bactericidal activity. The lowest tested concentration giving a reduction factor of at least 4 compared to the control was defined as the minimum bactericidal concentration (MBC).

All fungicidal tests were performed using Candida albicans (ATCC® 10231™). They were carried out in accordance with European standard EN 1275. A sample of the product was diluted with water and a test suspension of yeast cells was added. The number of yeast cells in the suspension was adjusted between 1.5 and 5.0×10 6 CFU/mL. The mixture was maintained at 20 ± 1 °C for 15 min ± 10 s. At the end of this contact time, an aliquot was taken; the fungicidal activity in this portion was immediately neutralized. The number of surviving yeasts in each sample was determined. Samples were serially diluted from 10 -1 to 10 -6 and each dilution was plated in duplicate on Sabouraud dextrose agar. After 48 h incubation of the plates at 37 °C, the colony-forming units par millimeter (CFU/mL) were counted. The fungicidal activity was determined by the difference between the logarithmic number of CFU/mL in the suspension at the beginning of the contact time (control test) and the logarithmic number of survivors per mL. This difference is presented as a reduction factor including its 95% confidence interval. A reduction factor of ≥4 was regarded as evidence of sufficient fungicidal activity according to EN 1275. The lowest concentration giving a reduction in virus titer of ≥4-log 10 was the minimum virucidal concentration (MVC).

Virucides, that attack and inactivate viral particles outside the cell (virions) by damaging their envelopes (and/or capsids) or genomes, are widely used to disinfect hard surfaces. This practice is useful for the prevention viral respiratory illnesses (e.g. CoViD-19, RSV, etc.) in households and community settings. Indeed, RSV is an enveloped RNA virus that primarily infects human epithelial cells within the nasopharynx. Infection with RSV is generally exhibited as lower respiratory tract disease, pneumonia, bronchiolitis or tracheobronchitis (Tang and Crowe, 2007) . Although all individuals can be infected, those at high risk include premature infants, young children, elderly, immunocompromised, and children under age 2 with chronic lung conditions. For these patients, RSV can cause pneumonia, leading to severe respiratory illness requiring hospitalization and causing sometimes death (Shi et al., 2020) .

For instance, within USA, 100,000 hospitalizations and 4,500 deaths annually are attributed to RSV infections (Tang and Crowe, 2007) . The treatment of RSV disease is essentially limited to supportive care such as oxygen therapy. Indeed, to treat severe RSV infections ribavirin is used but recent studies suggest that its use produces no benefit (Tang and Crowe, 2007) . It is noteworthy that RSV is also a major cause of nosocomial infections. If infection among healthy and immunocompetent individuals tends to be less severe, some specific factors may predispose to RSV infection include: crowding, exposure to tobacco and smoke, low socioeconomic status, and family history of atopy and asthma. Note that these factors are similar to those of SARS-CoV-2 infections (Zhang and Liu, 2020) . In contrast, RSV is more vulnerable than SARS-CoV-2 to environmental changes, particularly temperature and humidity: it loses up to 90% infectivity at room temperature after 48 hours (Tang and Crowe, 2007) . However, it may survive 3 to 30 hours on nonporous surfaces at room temperature (Tang and Crowe, 2007) . To prevent RSV infections, disinfectants based on sodium hypochlorite (1%), formaldehyde (18.5 g/L), glutaraldehyde (2%), iodine (1%), sodium deoxycholate (0.1%), sodium dodecyl sulphate and Triton X-100 can be used (Tang and Crowe, 2007; World Health Organization, 1993) . On the other hand, human CoV are susceptible to sodium hypochlorite (0.1%), organochlorine (0.1%), iodine (10%), ethanol (70%) and glutaraldehyde (2%) but more or less resistant to quaternary ammonium compounds according to their structures (Sattar et al., 1989) . For instance, [DiC 10 ][Cl] was able to reduce the viral loading of canine CoV by > 4.0 log 10 but benzalkonium chloride only by 3.0 log 10 despite the fact that both agents caused significant morphological damage to the virus (Pratelli, 2007) . Therefore, Schrank et al. (2020) claimed that: "this pandemic serves as an opportunity for enhanced antiseptics, and more specifically quaternary ammonium compounds development, as commercially available disinfectants have room for improvement both with formulation and concentration as well as effectiveness against both viral and bacterial contagions". As, some ethoxylated surfactants (e.g. Triton X-100, Nonoxynol-9 or Brij-97) have been shown to exhibit a virucidal activity due to their ability to solubilize the viral envelope of Epstein-Barr or herpes simplex viruses (Qualtiere and Pearson, 1979; Asculai et al., 1978) , and, as, CDs "are able to participate in the attack of viruses, and specifically SARS-CoV-2, in a large range of different ways" (Garrido et al., 2020) , we have investigated [DiC 10 ][Cl]/C 12 E 8 /CD ternary systems to obtain synergistic effects against enveloped viruses such as RSV instead of hazardous SARS-CoV-2.

In this context, we first determined the virucidal activity of [DiC 10 ][Cl] (Q), C 12 E 8 (E), and CD (,  or ) mixtures against RSV (Table 1) . Secondly, some physicochemical properties of these mixtures were recorded to establish the mechanism of action of these mixtures ( Table   2) . As control experiments, we evaluated the virucidal activity of each compound alone or in binary mixture against RSV at various concentrations. It is noteworthy that a reduction in virus titer of ≥4-log 10 (corresponding to an inactivation of ≥99.99%) was required to claim a Table 2 ). As previously reported this observation supports the insertion of free [DiC 10 ] cation within the viral envelope leading to membrane disorganization until destruction and virus inactivation . The opposite holds for C 12 E 8 : the MVC value is higher than the CMC of the surfactant (125 vs. 100 µM; see Table 2 ) which suggests that the C 12 E 8 micelles are the active species (Nardello-Rataj and Leclercq, 2016) . It is noteworthy that the hydrodynamic diameter (D h ) of the micelle was estimated to be around ~8 nm while the diameter of intact RSV was found at ~200 nm (see Table 2 ; Utley et al., 2008) . The adsorption of micelles on the viral envelope leads to the C 12 E 8 insertion in the outer membrane associated with a rapid flip-flop across the lipid membrane leading to the extraction of lipids above the CMC. In contrast, native CDs are not able to induce a virucidal activity against RSV at low concentrations (<1,000 μM) after 15 min or 2 h of contact time.

However, it is noteworthy that a reduction in virus titer of ≥4-log 10 after 24h was obtained for -CD at 6,000 µM. This result is close to the value observed against HSV-1 (Wallace et al., 2003 and C 12 E 8 due to the competitive inclusion of the surfactants into the CD cavity (Table 2) .

However, even if CDs are known to bind surfactant molecules below the CMC, no interactions of CDs with the surfactant micelles (above the CMC) were observed in this work (Nardello-Rataj and Leclercq, 2016) . Indeed, the hydrodynamic diameter (see D h in Table 2) of the micelles remains unchanged with or without CDs. In addition, for [DiC 10 ][Cl], the potentials as well as the degrees of ionization of micelle () were not influenced by the presence of CDs (Table 2 ). These findings confirm that both inclusion complexes and CDs remain in solution and are not in the micelle shell. Since the virucidal activity of [DiC 10 ] [Cl] or C 12 E 8 can be potentiated, the synergy index (SI) was estimated using the following equation:

where [X] is the concentration of X component in the mixture that produced a virucidal action (i.e. a reduction in virus titer of ≥4-log 10 ) and MVC X is the MVC observed for X acting alone (X = Q, E or CD). It is noteworthy that for CD, the [CD]/MVC CD is negligible with respect to the others as MVC CD is very high and [CD] very weak (see above and Table 1 ). A value of SI < 1 indicates a synergistic effect while a value SI > 1 means an antagonist effect (Zwart Voorspuij and Nass, 1957) . Finally, when SI = 0, simple additivity is observed between the components. As presented in Tables 1 and 2 , for all the [DiC 10 ][Cl]/CD mixtures, the MVCs are lower than the CMCs suggesting that the micelles are inactive species. Furthermore, as the CD affinity for solubilizing lipids are in the order -CD < -CD < -CD for phospholipids and -CD < -CD < -CD for cholesterol (Ohtani et al., 1989) Table 2 ) is in order -CD << -CD < -CD which is in perfect agreement with the virucidal efficiency (-CD < -CD < -CD, compare Tables 1  and 2 ). This observation supports the mechanism based on the micellar extraction of lipids from the viral envelope. Table 2 ). This observation supports that the co-micellization is governed by the C 12 E 8 molecules. Secondly, the -potential of comicelles was weaker than that of the [DiC 10 ][Cl] micelles whereas the degree of co-micelle ionization () was higher. These findings are due to the "podand" effect of the polyoxyethylene chains which can be seen as an open-chain equivalent to a crown ether able to complex [DiC 10 ] cations (Rauwel et al., 2012) . This cation binding podand decreases the electrostatic repulsion between the [DiC 10 ] cations, takes the chloride ions away and stabilizes the co-micelles. Based on these observations, we can suppose a relationship between the potential of the micelles and the virucidal activity. Indeed, the high -potential of co-micelles compared to pure C 12 E 8 micelles improves the probability of the attachment of [DiC 10 ] cations to the anionic regions (i.e. phospholipids and proteins) prior to adsorption inside the envelope (Tobe et al., 2015) . It is believed that increasing the appearance of the cationic region of [DiC 10 ] cations in mixed micelles will increase the adsorption of [DiC 10 ] cations on the anionic region of the viruses. This behavior associated with the weak CMC of the mixed system is supposed to be responsible for the observed synergistic effect. (2) the -CD is more expensive than the -CD. As the sizes of the comicelles as well as the -potentials and the degrees of ionization of micelle () were not influenced by the presence of CDs (see Table 2 It is noteworthy that a concomitantly mechanism in which free [DiC 10 ] cations interact, penetrate and weaken the viral envelope due to the multiple equilibria in [DiC 10 ][Cl]/C 12 E 8 /CD ternary systems also take place (see Figure 2 ). Indeed, next to the 

To extend the scope of the systems, the biocidal (or virucidal) action against DNA enveloped viruses, bacteria and fungus was carried out under similar conditions (Table 3) .

The two selected lipid-containing DNA viruses are the well-known HSV-1 and VACV. The HSV viruses lead to orofacial, ophthalmic and genital herpes, and sometimes to encephalitis whereas the VACV is used as model infections for human smallpox caused by the variola virus (VARV). As most of bacteria are Gram-negative, we worked with Pseudomonas aeruginosa an archetypal encapsulated rod-shaped pathogen that can cause disease in plants and animals, including humans. On the other hand, a commensal fungus which lives in the human mouth and gastrointestinal tract such as Candida albicans has been investigated because their overgrowth results in candidiasis that results in septicemia (Leclercq et al., 2020) . It is noteworthy that according to the well-known disinfection scale, the pathogen susceptibility to chemical biocides is in the following order: enveloped viruses > bacteria > fungus > non-enveloped viruses (Prince and Prince, 2001) .

For each pathogen, the biocidal (or virucidal) activity of [DiC 10 ][Cl], C 12 E 8 and CDs alone or in equimolar mixture has been determined (Table 3) (Tables 2 and 3) .

Insert Table 3 .

This differential susceptibility is correlated with the virus size: the diameters are ~120, ~200

and ~320 nm for HSV-1, RSV and VACV (Salmon et al., 1998; Utley et al., 2008; Malkin et al., 2003) . Indeed, based on the proposed virucidal mechanism (see above), [DiC 10 ]/C 12 E 8 co-micelles and [DiC 10 ] alone can interact and alter the viral envelope, after the insertion of [DiC 10 ] cations, facilitating the lipid extraction by the CDs and leads to virus inactivation. We can therefore suppose that the required amount of inserted [DiC 10 ] cations to obtain a sufficient disorganization of the membrane prior to virus inactivation is higher for VACV than RSV or HSV-1 because of the viral size.

As expected, the C 12 E 8 or CDs alone were inactive against P. aeruginosa and C. albicans, Therefore, only additive responses were obtained for binary and ternary mixtures (SI = 1, Influenza A2 and VACV), and fungi (e.g. C. albicans and T. mentagrophytes). As depicted in Tables 1 and 3 Moreover, the efficiency against SARS-CoV-2 requires probably higher concentration in active ingredients. Indeed, in the Bardac 205M family only the products containing at least 

We have demonstrated that [DiC 10 ] cations associated with C 12 E 8 and native CDs show synergistic action against enveloped viruses (RSV, HSV-1 and VACV) and fungi (C. Modification of lipid interactions allows the virus inactivation or cellular death by the exposure of the genome. As this synergistic effect, based on complex interactions and multiple equilibria, provides broad-spectrum eradication (enveloped viruses, bacteria and fungi), it is unlikely to lead to the development of pathogens resistance against these three components mixtures. These highly relevant results, in the current pandemic situation, support that this synergistic effect provides elements valuable for prevention of respiratory illnesses due to enveloped viruses, including SARS-CoV-2, and other hazardous pathogens in households and community settings. Work is under way to extend the concept of these systems to other disinfectants.

The manuscript was written through contributions of all authors who approve the final version of the manuscript. or virucidal activity in log 10 titer reduction factor recorded at room temperature after 15 min of contact time for HSV-1 (1.5×10 7 TCID 50 /mL), VACV (1.1×10 7 TCID 50 /mL), C. albicans (1.6×10 6 CFU/mL) and 5 min for P. aeruginosa (1.0×10 8 CFU/mL). (*) Minimum virucidal concentration (MVC) = the lowest concentration able to inactivate at least 99.99% of viruses. b Calculated according equation 1.

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Figure 1. Effects of didecyldimethylammonium cation

C 12 E 8 /CD mixtures against RSV: (i) adsorption of [DiC 10 ]/C 12 E 8 co-micelles through electrostatic interaction with the viral envelope, (ii) insertion of

Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur et de la Recherche, Région Hauts-de-France and Fonds Européen de Développement Régional (FEDER) are acknowledged for supporting and funding partially this work. We are most grateful to Gaétan Rauwel (Laboratoires ANIOS, France) and Anny Dewilde (Université de Lille, Laboratoire de Virologie, France).