Sulfonated Nanomaterials with Broad-Spectrum Antiviral Activity Extending beyond Heparan Sulfate-Dependent Viruses

Viral infections are among the main causes of death worldwide, and we lack antivirals for the majority of viruses. Heparin-like sulfated or sulfonated compounds have been known for decades for their ability to prevent infection by heparan sulfate proteoglycan (HSPG)-dependent viruses but only in a reversible way. We have previously shown that gold nanoparticles and β-cyclodextrins coated with mercapto-undecane sulfonic acid (MUS) inhibit HSPG-dependent viruses irreversibly while retaining the low-toxicity profile of most heparin-like compounds.

ABSTRACT

Viral infections are among the main causes of death worldwide, and we lack antivirals for the majority of viruses. Heparin-like sulfated or sulfonated compounds have been known for decades for their ability to prevent infection by heparan sulfate proteoglycan (HSPG)-dependent viruses but only in a reversible way. We have previously shown that gold nanoparticles and β-cyclodextrins coated with mercapto-undecane sulfonic acid (MUS) inhibit HSPG-dependent viruses irreversibly while retaining the low-toxicity profile of most heparin-like compounds. In this work, we show that, in stark contrast to heparin, these compounds also inhibit different strains of influenza virus and vesicular stomatitis virus (VSV), which do not bind HSPG. The antiviral action is virucidal and irreversible for influenza A virus (H1N1), while for VSV, there is a reversible inhibition of viral attachment to the cell. These results further broaden the spectrum of activity of MUS-coated gold nanoparticles and β-cyclodextrins.

INTRODUCTION

In the absence of vaccines, antivirals represent a major line of defense against viruses. However, currently available antivirals are virus specific, and they have been developed against only a limited number of viruses. The recent pandemic of COVID-19 calls our attention to the major benefit of identifying broad-spectrum antivirals, ready to use when a new epidemic starts.

In the past years, we worked on developing broad-spectrum antiviral compounds. To potentially maximize their efficacy in vivo, we sought to develop compounds that had an irreversible inhibition mechanism (i.e., virucidal compounds). Irreversible inhibition is insensitive to dilution, for instance, in body fluids, and thus does not require a constant concentration of the inhibitors in vivo. To achieve a broad-spectrum response, we designed compounds mimicking heparan sulfate proteoglycan (HSPG), a receptor broadly present on eukaryotic cells and used by a wide variety of viruses (here called HSPG-dependent viruses) to attach to host cells and subsequently find a more specific entry receptor (1). This approach, which began with seminal studies on heparin, is known to lead to efficient in vitro inhibition of many viral strains although with a reversible mechanism of action. To achieve irreversible inhibition, we added to the design long hydrophobic linkers conferring to the compounds the ability to permanently inhibit a large number of HSPG-dependent viruses (2, 3). The key chemical moiety used was a long hydrophobic chain terminating with a sulfonic acid (undecyl sodium sulfonate) presented in a multivalent way, in order to achieve a good inhibitory concentration and strong hydrophobic contact (required to render the interaction irreversible). In the first designed compound (named MUS:OT NP) (Fig. 1a), such a moiety was fixed on gold nanoparticles (4-nm gold nanoparticles [NPs] coated with a mixture of octane thiol [OT] and mercapto-undecane sulfonic acid [MUS]) (2). In the second compound (named CD1), the moiety was linked to the primary face of β-cyclodextrins (CD) (3) (Fig. 1b).

FIG 1.

(a) Chemical structure of MUS:OT NP, composed of a gold (Au) core and the ligands mercapto-undecansulfonate (MUS) and 1-octanethiol (OT). (b) Chemical structure of CD1 β-cyclodextrin (in blue) conjugated with MUS.

Both compounds achieved virucidal action against viruses binding to HSPG, such as herpes simplex virus 1 (HSV-1), HSV-2, respiratory syncytial virus (RSV), dengue virus, and many other viruses. Both compounds also exhibited good ex vivo and in vivo efficacy (2, 3). We have thus shown that the use of undecyl sodium sulfonate (multivalently presented) generates compounds that display the same broad-spectrum antiviral and low-toxicity profile of heparin but, unlike heparin, can exert irreversible and virucidal activity.

In this study, we investigate the interaction of our previously reported compounds (MUS:OT NP and CD1) with viruses that do not use HSPGs as attachment receptors (here called HSPG-independent viruses), such as different strains of influenza virus (4) and vesicular stomatitis virus (VSV) (5, 6). We highlight the virucidal activity of both compounds against influenza virus and virustatic activity against VSV. We further show that the activity against VSV is partially cell mediated but mostly caused by interference with viral attachment.

RESULTS

Antiviral activities of MUS:OT NP and CD1 against HSPG-independent viruses.

Antiviral assays were carried out by incubating different strains of influenza virus or VSV with a dose range of MUS:OT NP or CD1, whose structure is shown in Fig. 1, for 1 h at 37°C before infection of appropriate cell lines. The results are presented in Table 1. Both compounds display inhibitory activity in the absence of toxicity, with 50% effective concentrations (EC₅₀s) generally lower for MUS:OT NP than for CD1, in line with our previous publications (2, 3). Importantly, we also verified that MUS:OT NP inhibits the same viruses as CD1, which were not tested with this compound in our previous works (see Table S1 in the supplemental material), confirming the same spectrum of activity of both materials.

TABLE 1.

Antiviral activities of MUS:OT NP and CD1 against HSPG-independent viruses^a

Compound	Virus	EC50 (μg/ml) (95% CI)	EC50 (μM)	CC50 (μg/ml)
MUS:OT-NP	VSV	0.053 (0.032–0.084)	0.000241	>300
	A/Netherlands/602/2009 (H1N1)	1.38 (1.17–1.62)	0.00627	>300
	A/Singapore/37/2004 (H3N2)	12.0 (8.59–16.7)	0.0545	>300
	A/Vietnam/1203/2004 (H5N1)	4.01 (3.15–5.02)	0.0182	>300
	B/Wisconsin/01/2010	7.08 (3.73–12.8)	0.0322	>300
CD1	VSV	45.4 (35.8–57.6)	15.8	>300
	A/Netherlands/602/2009 (H1N1)	6.28 (4.58–8.39)	2.17	>300
	A/Singapore/37/2004 (H3N2)	17.3 (13.7–22.0)	6.01	>300
	A/Vietnam/1203/2004 (H5N1)	53.2 (28.2–149)	18.5	>300
	B/Wisconsin/01/2010	13.1 (10.0–17.5)	4.55	>300

^aEC₅₀, 50% effective concentration (50% maximal effect); CI, confidence interval; CC₅₀ cytotoxic concentration causing 50% cell death.

Antiviral activities of other heparan sulfate-mimicking compounds against HSPG-independent viruses.

To confirm the HSPG dependence of VSV and influenza, we further investigated the inhibitory activity of heparin, ι-carrageenan, and K5NOS(H) (1), other heparan sulfate analogues previously shown to inhibit HSPG-dependent viruses. To this end, we used the same protocol as the one described above, i.e., preincubation of viruses and the compound for 1 h at 37°C before addition to cells. The results, presented in Fig. 2a and b, show different inhibition profiles of the two viruses. H1N1 is inhibited by ι-carrageenan and K5NOS(H) but very weakly inhibited by heparin and only at the highest concentration tested (Fig. 2a). Conversely, VSV is not inhibited by any of the sulfate analogues tested (Fig. 2b).

FIG 2.

(a and b) Antiviral activity of heparan sulfate analogues against H1N1 A/Netherlands/602/2009 (a) and VSV (b). Viruses and compounds were incubated for 1 h at 37°C and subsequently added to cells. Infectivity was evaluated at 24 hpi for both viruses. The percentage of infection was calculated by comparing the number of infected cells for H1N1 or plaques for VSV to those for the untreated controls. (c and d) Virucidal activities of MUS:OT NP, CD1, and K5NOS(H) against H1N1 (c) and MUS:OT NP and CD1 against VSV (d). Approximately 10⁵ focus-forming units (ffu) (H1N1) or ∼10⁵ PFU (VSV) were incubated for 1 h at 37°C with the indicated concentrations of the compound and subsequently serially diluted in cells. Infectious titers were evaluated under each treatment condition at dilutions at which the concentration of compound is not active. Results are expressed as means and standard errors of the means (SEM) from three independent experiments performed in duplicate. Statistical significance relative to the untreated control was calculated using one-way ANOVA (**, P < 0.01; *, P < 0.05). EC₅₀, 50% effective inhibitory concentration.

Virucidal activities of MUS:OT NP and CD1 against VSV and influenza virus.

Next, we investigated if the compounds show virucidal activity, i.e., irreversible inhibition, against VSV and H1N1. This property was previously reported to be exerted by the nanomaterials against HSPG-dependent viruses (1–3). The virucidal assay was performed as previously reported (2), by incubating the virus with the inhibitory concentration of the compound, followed by serial dilutions of the complex and evaluation of residual viral infectivity at concentrations at which the compound is not active.

The results revealed the virucidal activities of both compounds against influenza virus, with reductions of 2.4 logs at 300 μg/ml and 1.6 logs at 100 μg/ml for MUS:OT NP and of 1.0 log at 300 μg/ml for CD1 (Fig. 2c). The extent of virucidal inhibition is comparable to that observed for RSV previously (2, 3). Importantly, we also included in the virucidal assay a highly sulfated polysaccharide, K5NOS(H), previously reported to exert antiviral activity against multiple HSPG-dependent viruses. As shown in Fig. 2c, K5NOS(H) is not virucidal against influenza virus despite displaying good antiviral activity (Fig. 2a). These results confirm that the presence of the long alkylic chains is a key factor in the virucidal mechanism of action (3). Conversely, the two compounds do not display virucidal activity against VSV (Fig. 2d) despite the low EC₅₀ measured in inhibitory assays (Table 1).

Mechanisms of action of MUS:OT NP and CD1 against VSV.

As the two compounds show different activity profiles against VSV and influenza virus, i.e., virustatic versus virucidal activity, we carried out time-of-addition experiments, flow cytometry, and immunofluorescence analyses to elucidate the antiviral mechanism of action against this virus.

CD1 (Fig. 3a) or MUS:OT NP (Fig. 3b) was either (i) preincubated for 1 h at 37°C with the virus (pretreatment with virus plus infection at 37°C), (ii) preincubated for 2 h at 37°C with the cells and then washed out before infection (pretreatment of cells), (iii) directly added to the cells together with the virus at 37°C (cotreatment), or (iv) preincubated with the virus for 1 h at 37°C followed by the addition of the mixture of the virus and compound on prechilled cells for 1 h at 4°C to prevent viral entry (pretreatment with virus plus infection at 4°C). Under all conditions, after washing out the viral inoculum, the cells were overlaid with medium containing methylcellulose and incubated at 37°C for 24 h. Dose-response curves and EC₅₀s were not significantly different under all conditions for CD1, except when the cells were pretreated with the compound, where no activity was observed (Fig. 3a). Similarly, inhibitory activities under pre- and cotreatment conditions were comparable for MUS:OT NP (Fig. 3b). Interestingly, pretreatment of cells with MUS:OT NP also inhibited VSV infection but with an EC₅₀ significantly higher than that reported upon pre- or cotreatment with the virus (2.6 versus 0.053 μg/ml). Of note, a similar assay performed with HSV-2, which binds HSPG moieties, showed no inhibition (Fig. S3). The partial cell-mediated inhibition observed against VSV is thus virus specific. To further dissect the contribution of cell-mediated inhibition versus direct virus inhibition, we infected Vero cells with green fluorescent protein (GFP)-expressing VSV (multiplicity of infection [MOI] of 5) and measured GFP expression by flow cytometry at early times postinfection, in the presence or absence of MUS:OT NP added for pretreatment of cells or cotreatment. The results revealed significantly greater inhibition with cotreatment (Fig. 3c), confirming that the major component of inhibition is a direct interaction with the virus. To highlight the step of the viral life cycle inhibited by the compound, we performed a cotreatment assay (MOI of 20) and fixed the cells 1 h after incubation at 37°C for immunostaining with an anti-VSV antibody. In contrast to the untreated virus, almost no signal was observed in the presence of the compound, indicating that MUS:OT NP prevents the virus from binding and/or entering the cell (Fig. 3d). Finally, the inhibitory activity was lost if infection was preceded by a 1-h incubation at 4°C (Fig. 3b), in contrast to what was observed for HSV-2 (Fig. S3). This confirms that the association of the virus with the NP is reversible and likely lost at 4°C.

FIG 3.

(a and b) Mechanisms of action of CD1 (a) and MUS:OT NP (b) against VSV. The compounds were either added to cells for 2 h before infection (pretreatment of cells), incubated with the virus for 1 h and then added to cells at 37°C (pretreatment with virus infection at 37°C), added with the virus to the cells (cotreatment), or preincubated with the virus for 1 h and then added to cells at 4°C for 1 h before shifting to 37°C (pretreatment with virus infection at 4°C). Infectivity was evaluated at 24 hpi. (c) Flow cytometry was performed at 3 hpi with Vero cells infected with VSV expressing GFP (MOI of 5) after pretreatment with MUS:OT NP for 2 h (pretreated cells) or in the presence of MUS:OT NP (cotreatment). Percentages of infection are normalized according to the untreated (UT) conditions. (d) Vero cells that were uninfected (mock) or infected with VSV (MOI of 20) in the absence (UT) or presence of MUS:OT NP (100 μg/ml) were fixed at 1 hpi and subjected to immunostaining with an anti-VSV polyclonal antibody. Green, VSV; red, Evans blue; blue, DAPI. Bars, 10 μm. Results are expressed as means and SEM from two independent experiments performed in duplicate. Statistical significance relative to the untreated control was calculated using one-way ANOVA (****, P < 0.001).

DISCUSSION

Influenza viruses are known to attach to sialic acid to infect cells (4). However, we show that recently developed HSPG-mimicking compounds (MUS:OT NP and CD1 [2, 3]) are able to inhibit several influenza A (H1N1, H3N2, and H5N1) and B virus strains. Our results are supported by glycan array studies that highlight interactions between the influenza virus hemagglutinin protein and sulfated glycans (particularly with a sulfation on N-acetylglucosamine, linked to galactose and sialic acid) (7). In line with the glycan array data, the lowest EC₅₀s achieved with our HSPG-mimicking compounds were obtained against the H1N1 strain, which was also reported to have the highest affinity for sulfated glycans (7). In addition, we demonstrate that H1N1 is also inhibited by highly sulfated molecules, such as ι-carrageenan, in line with previous literature (8), and by K5NOS(H), due to the affinity of hemagglutinin for sulfated and sulfonated molecules. Our results also provide evidence of full or partial virucidal activity against influenza virus of MUS:OT NPs or CD1, respectively. Altogether, these data suggest a direct interaction between the virus and the compound and subsequent virucidal activity mediated by the hydrophobic portion of both MUS:OT NPs and CD1. Importantly, despite good inhibitory activity, K5NOS(H) did not exert any virucidal effect, further supporting the importance of a long hydrophobic linker to mediate virucidal activity (2, 3). Of note, in contrast to our previous observations (3), in the present study, we report that CD1 inhibits the infectivity of H3N2. An optimized synthesis protocol as well as a different number of ligands per β-cyclodextrin likely account for the broader activity of the recently synthesized CD1 molecules.

VSV is also inhibited by MUS:OT NPs and CD1 (Fig. 3b) but only in a reversible way, as shown by the lack of inhibition when the compound-virus complex is diluted out before infection (Fig. 2d). Unlike influenza virus, VSV is not inhibited by any of the heparin analogues. Importantly, these results clearly show that the virucidal activity is a feature independent of the EC₅₀ value since the EC₅₀ of MUS:OT NP against VSV is lower than that measured against H1N1 (Table 1), but the interaction is irreversible only against the latter. Interestingly, both CD1 and MUS:OT NP show maximal inhibition upon direct interaction with VSV, as observed for HSV-2. This suggests that the types of interaction in both cases must be similar, although the action on HSV-2 is stronger and irreversible due to the natural dependence of the virus on HSPG (2) (see Fig. S3 in the supplemental material).

Of note, CD1 and MUS:OT NP do not show comparable activity profiles against VSV. CD1 activity is maintained under all experimental conditions (pretreatment, cotreatment, and pretreatment plus 1 h at 4°C) except cell pretreatment. These results suggest an electrostatic interaction that does not play a role when CD1 is preincubated with the cell and washed out before the addition of the virus. Our interpretation is that viral inhibition depends on a rather quick interaction of CD1 with the virus that is sufficient to prevent viral attachment. Interestingly, under the same experimental conditions, part of the inhibition is maintained for MUS:OT NP, indicating a partial cell-mediated effect for this compound. This effect is not observed with HSV-2 and is thus virus specific. In contrast to CD-based molecules, NPs are able to interact with or cross cellular membranes (9). This cell-mediated inhibition observed in the case of VSV is probably due to interactions with specific domains of the cellular membranes or internalization of the particles during pretreatment of the cells and subsequent colocalization with the virus in cytosolic compartments, as previously described for other compounds (10). Finally, we demonstrate that the nanoparticles lose their inhibitory ability when added to the cells together with the virus and incubated at 4°C for 1 h. In line with their virustatic effect, we assume that at this temperature, the nanoparticle-virus complex dissociates.

Conclusions.

We previously demonstrated that irreversible inhibition of viral infectivity of HSPG-dependent viruses is possible with heparin-mimicking compounds presenting undecyl sulfonic acids in a multivalent fashion. Here, we show that these molecules also exert the same effect also against several sialic acid-dependent influenza virus strains. In line with glycan array results (7), we propose that this interaction is due to the ability of hemagglutinin of influenza virus to bind sulfated sugars. We further demonstrate that the same compounds can achieve reversible inhibition of other HSPG-independent viruses such as VSV. In this context, we highlight complex interaction profiles that are consistent with a weak electrostatic interaction. In light of these results, further investigations will be directed to an even broader panel of viruses in order to define structural commonalities and better understand the mechanism of action of our sulfonated nanomaterials against HSPG-independent viruses.

MATERIALS AND METHODS

Reagents.

Heparin from porcine mucosa (catalogue number H4784) and ι-carrageenan (catalogue number C1138) were purchased from Sigma, and K5NOS(H) was synthesized by Glycores SRL (Milan, Italy) (11). Heparin and K5NOS(H) were resuspended in water, aliquoted, and kept at −20°C.

Nanoparticles and cyclodextrins.

MUS:OT NPs, gold nanoparticles covered with a binary shell of octane thiol (OT) and 11-mercapto-undecansulfonate (MUS), were synthesized as reported previously (12). The characterization is shown in Fig. S1 in the supplemental material. CD1, composed of β-cyclodextrins bearing MUS, was synthesized as reported previously (3). The characterization is shown in Fig. S2.

Cells lines.

A549, LLCMK2, Madin-Darby canine kidney (MDCK), and Vero cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (Sigma-Aldrich) at 37°C in an atmosphere of 5% CO₂.

Viruses.

Influenza A/Netherlands/602/2009 (H1N1) (Pdm09), A/Singapore/37/2004 (H3N2), A/Vietnam/1203/2004 (H5N1), and B/Wisconsin/01/2010 viruses were a gift from M. Schmolke (University of Geneva, Switzerland), and they were propagated on MDCK cells in DMEM supplemented with l-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)–trypsin (0.2 μg/ml) and titrated on MDCK cells with an indirect immunoperoxidase staining procedure using an anti-influenza A virus (IAV) monoclonal antibody (MAB5001; Millipore) and an anti-influenza B virus monoclonal antibody (MAB5002; Millipore). Human metapneumovirus (HMPV) (ATCC) was propagated in Vero cells in DMEM supplemented with 1% penicillin-streptomycin and trypsin (200 ng/ml) and titrated by the indirect immunoperoxidase staining procedure using an anti-HMPV monoclonal antibody (HMPV 24; Bio-Rad). Parainfluenza virus 3 (PIV3) (ATCC) was propagated in LLCMK2 cells in DMEM supplemented with 1% penicillin-streptomycin and trypsin (200 ng/ml) and titrated by a plaque assay. Recombinant VSV expressing GFP (13) was a gift from D. Garcin (University of Geneva, Switzerland), and it was propagated on Vero cells and titrated by a plaque assay. Zika virus (ZIKV) strain PRVABC59 was provided by M. Alves (University of Bern, Switzerland).

Toxicity assay.

Cells were seeded in a 96-well plate. Compounds were serially diluted (1:3) in medium starting from 300 μg/ml for all the cell lines, with the exception of Huh7.5 cells, in which 100 μg/ml was the highest concentration tested, and added to cells for a time equal to that for the antiviral assay (i.e., 24 h of incubation for Vero cells, the cell line used for VSV, and 24 h for MDCK cells, the cell line used for IAV). At the end of the incubation period, cells were washed, a thiazolyl blue tetrazolium bromide solution (0.5 mg/ml) in medium was added to cells for 3 h at 37°C, the cells were subsequently lysed with pure dimethyl sulfoxide (DMSO), and the absorbance was read at 570 nm. Percentages of viability were calculated by comparing the absorbance in treated wells to that in wells treated with water with equal volumes of the drugs. The 50% cytotoxic concentration (CC₅₀) values were calculated with Prism 8 (GraphPad, USA).

Inhibition assay for HCV.

Hepatitis C virus (HCV) Jc1 particles were produced by electroporating Huh7.5 cells with chimeric JFH1-J6/C3 full-length RNA generated as previously described (14), and the 50% tissue culture infective dose (TCID₅₀) per milliliter was calculated using a previously reported protocol (15). Viral particles (MOI of 2) were incubated with decreasing concentrations of MUS:OT NPs (100, 20, 4, 0.8, and 0 μg/ml) for 1 h at 37°C. Huh7.5 cell plated at 25,000 cells/well in a 24-well plate were infected with the virus-NP mixture. At 6 h postinfection (hpi), cells were washed, and the culture medium was changed. At 48 h, total intracellular RNA was extracted using the RNeasy minikit (Qiagen), and the relative number of intracellular HCV RNA copies was assessed by reverse transcription-quantitative PCR (RT-qPCR) as described previously (16).

Inhibition assay for HMPV.

A549 cells (at 8,000 cells per well) were seeded in a 96-well plate. MUS:OT NPs were serially diluted (starting from 300 μg/ml with 1:3 dilutions) in medium and incubated with virus (MOI of 0.01) for 1 h at 37°C, and the mixtures of the compound and virus were then added to the cells to allow viral adsorption for 3 h at 37°C. The monolayers were then washed and overlaid with 1.2% methylcellulose medium containing trypsin (200 ng/ml). Three days after infection, cells were fixed with cold methanol and acetone for 1 min and subjected to HMPV-specific immunostaining. Immunostained plaques were counted, and the percent inhibition of virus infectivity was determined by comparing the number of plaques in treated wells with the number in untreated control wells.

Inhibition assay for PIV3.

LLCMK2 cells (at 80,000 cells per well) were seeded in a 24-well plate. MUS:OT NPs were serially diluted (starting from 300 μg/ml with 1:3 dilutions) in medium and incubated with virus (MOI of 0.01) for 1 h at 37°C, and the mixtures of the compound and virus were then added to the cells to allow viral adsorption for 2 h at 37°C. The monolayers were then washed and overlaid with 0.8% methylcellulose medium containing trypsin (200 ng/ml). Two days after infection, cells were fixed with ethanol and stained with crystal violet. Plaques were counted, and the percent inhibition of virus infectivity was determined by comparing the number of plaques in treated wells with the number in untreated control wells.

Inhibition assay for influenza viruses.

MDCK cells (13,000 cells per well) were seeded in a 96-well plate. MUS:OT NPs, CD1, and heparan sulfate mimetics were serially diluted (starting from 300 μg/ml with 1:3 dilutions for H1N1 and from 100 μg/ml for the other strains) in DMEM and incubated with virus (MOI of 0.01) for 1 h at 37°C, and the mixtures of the compound and virus were then added to the cells to allow viral adsorption for 1 h at 37°C. The monolayers were then washed and overlaid with DMEM. Eighteen hours after infection, cells were fixed with methanol and subjected to influenza virus-specific immunostaining. Immunostained cells were counted, and the percent inhibition of virus infectivity was determined by comparing the number of plaques in treated wells with the number in untreated control wells.

Inhibition assay for VSV and ZIKV.

Vero cells (100,000 cells per well) were seeded in a 24-well plate. MUS:OT NPs and CD1 were serially diluted in DMEM (starting from 300 μg/ml with 1:3 dilutions) and incubated with virus (MOI of 0.01) for 1 h at 37°C, and the mixtures of the compound and virus were then added to the cells to allow viral adsorption for 1 h at 37°C. The monolayers were then washed and overlaid with 1.2% methylcellulose medium. One day after infection for VSV and 3 days after infection for ZIKV, cells were fixed with ethanol and stained with crystal violet. Plaques were counted, and the percent inhibition of virus infectivity was determined by comparing the number of plaques in treated wells with the number in untreated control wells.

Virucidal assay.

Viruses [10⁵ PFU for VSV and 10⁵ focus-forming units (FFU) for influenza virus A/Netherlands/602/2009 (H1N1)] and MUS:OT NPs or CD1 (300 μg/ml or 100 μg/ml) were incubated for 1 h at 37°C, and the virucidal effect was then investigated with serial dilutions of the mixtures. Viral titers were determined at dilutions at which the material was present at a residual concentration lower than the EC₅₀ previously determined (for VSV, dilutions of <1:10,000 for MUS:OT NP and <1:100 for CD1 were considered, while for influenza virus, dilutions of <1:1,000 for MUS:OT NP and <1:100 for CD1 were considered).

Time-of-addition experiments.

Nanomaterials were added to cells 2 h before infection (pretreatment of cells), preincubated with the virus for 1 h and then added to cells (pretreatment with virus plus infection at 37°C), or added directly with the virus to cells (cotreatment). For the pretreatment of cells, the nanomaterial was washed out before viral infection. Under all conditions, after 1 h of viral infection at 37°C (MOI of 0.005), cells were washed and overlaid with 0.8% methylcellulose medium. For attachment assays, MUS:OT NP was preincubated for 1 h with the virus and subsequently added to cells for 1 h at 4°C, followed by a wash and the addition of an overlay with 0.8% methylcellulose medium. At 24 hpi, cells were fixed and stained with crystal violet, and plaques were counted.

Immunofluorescence assay.

Anti-VSV primary antibody produced by a hybridoma cell line (1:100) (17) was a kind gift from Giulia Torriani. Alexa Fluor 488 goat anti-mouse (1:2,000) (Life Technologies) was used as a secondary antibody, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the cytoplasm was stained with Evans blue. Images were acquired with a Zeiss LSM 700 Meta confocal microscope.

Flow cytometry.

VSV-infected cells were washed, detached with trypsin at 0.05 mg/ml, resuspended in phosphate-buffered saline (PBS), centrifuged for 5 min at 3,000 rpm, and resuspended in a solution containing PBS, 1% paraformaldehyde, and 1% bovine serum albumin (BSA). Cell suspensions were analyzed with BD Accuri C6.

Statistical analysis.

Where possible, EC₅₀ values were calculated by regression analysis using the dose-response curves generated from the experimental data using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). The CC₅₀ was determined using logarithmic viability curves. One-way analysis of variance (ANOVA), followed by a Bonferroni test, was used to assess the statistical significance of the differences between treated and untreated samples, where appropriate. Significance was set at the 95% level. EC₅₀ values were compared using the sum-of-squares F test.