2.1. Test material
The test material was a universal cloth (Jemako®, Rhede, Germany) made of nonwoven microfibers (80% polyester/20% nylon). It was coated with salt at various concentrations and dried overnight at room temperature (20°C). It was stored under nitrogen atmosphere in a clean sealed plastic bag until use. Before being tested, the fabric was cut into 1 cm2 pieces (three pieces per treatment group) and placed in a sterile 24-well plate. Both sides of the fabric were exposed to ultraviolet C radiation by using a TUV 30W/G30 T8 lamp (Philips Lighting, Signify, Eindhoven, The Netherlands) for 30 min.
2.2. Coating solution
A salt solution containing 29.03% w/v NaCl in demineralized water (29.03 g/100 ml) and 1% Tween 20 (Merck Sigma Aldrich, Darmstadt, Germany) was used as the starting concentration (Quan et al. 2017). A five-fold dilution in demineralized water was also prepared.
2.3. Coating procedures
The salt solutions were applied to the test material by using two coating procedures that could be accessible in a home environment: spraying and dipping. For spray coating, a system consisting of a mini spray valve (Nordson EFD781) mounted on an automated robot (Janome JR2304) was used to control the amount of salt deposition with the following defined parameters: speed of deposition, 40 mm/s; distance between spray head and test materials, 40 mm; pressure on the cartridge containing the salt solution, 0.4 bar; and deposition pattern, straight parallel lines 4 mm apart. The valve aperture controlling the flow (stroke) was set at 3 or 5, resulting in salt concentrations of 2.13 and 11.03 mg/cm2 on the test fabrics labeled Spr S3 and Spr S5, respectively.
For dip coating, a five-fold diluted salt solution (5.81% NaCl; 0.2% Tween 20) was used with an automated dip coater (KSV Nima medium, Biolin Scientific). The automatic system allowed the controlled full immersion of the fabrics for 3 s followed by withdrawal at a constant speed of 100 mm/min. The fabric samples were then suspended vertically for 30 min to drain and dry before storage. The dip-treated test material was labeled Dip Dil5× with an estimated salt concentration of 10 mg/cm2.
Salt distribution and crystal aspect on the coated fabrics were checked via scanning electron microscopy (FEI Scios2, ThermoFisher Scientific, Waltham, MA, USA) in a low vacuum mode and energy-dispersive X-ray spectroscopy (X-Max 50 mm2 detector; Oxford Instruments, High Wycombe, UK; AZtec software control).
2.4. Epithelial tissues
Primary human nasal epithelial cultures from a pool of donors were provided by Epithelix Sàrl (MucilAir™ pool of donors, article reference EP02MP, Epithelix Sàrl, Genèva, 514 Switzerland) and handled in accordance with the manufacturer’s instructions. MucilAir™ is a 3D in vitro cell model of the human airway epithelium cultured at the air–liquid interface. These epithelia are fully mature and functional.
2.5. Generation of virus stock solution
We used SARS-CoV-2 that was synthetically constructed using a yeast cloning system (Thao et al. 2020). The full-length sequence of SARS-CoV-2 was confirmed by sequencing. SARS-CoV-2 was propagated in Vero E6 cells for 48 h, and, afterwards, supernatant was centrifuged at 500 × g for 5 min, aliquoted, and stored at − 80°C until it was used for the infection assays. The viral titer of the stock was determined via a 50% tissue culture infective dose (TCID50) assay on Vero E6 cells. A 96-well plate was seeded with 2.0 × 106 cells, i.e., 20,000 cells per well, 24 h before viral infection. The virus stock was serially diluted at 1:10 and incubated for 72 h. After incubation, the medium was removed, and the cells were fixed and stained with crystal violet. The titer was determined in accordance with the methods of Spearman–Kärber (Hierholzer and LKillington 1996). The virus titer in the produced viral stock resulted in a titer of 4.0 × 105 TCID50/ml. VeroE6 cells were cultured in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 15 mM HEPES (Gibco; Thermo Fisher Scientific).
2.6. Infection assay
SARS-CoV-2 virus stock (50 µl) with a titer of 4.0 × 105 TCID50/ml was exposed dropwise on the fabric at room temperature for 10 min. After exposure, the virus particles were retrieved from the mask material by adding 500 µl of MucilAir™ medium, mixing via a few pipetting movements, and incubating at room temperature for 5 min. The obtained resolved virus solutions were immediately used for infection assays on primary human nasal epithelial cultures, and the leftover of the virus suspension was stored at − 80°C for the subsequent titration and genome copy quantification.
Prior to infection, each insert was washed apically with the culture medium for 10 min. Three inserts were used per treatment group, i.e., 3 technical replicates. For inoculation, 200 µl of the virus-containing solution was applied to the apical side of the cultures and incubated at 33°C in a humidified incubator with 5% CO2 for 3 h. Subsequently, the inserts were washed rapidly and apically with the culture medium thrice to remove unbound virus particles.
For post-infection viral quantification, an apical wash with 200 µl of MucilAir™ culture media at 33°C for 20 min was collected after 3, 24, 72, and 144 h of incubation. The collected apical liquids were stored at − 80°C. Titration and virus genome copy number quantification were performed.
The following controls were included: virus control (20 µl of a virus stock of 4.0 × 105 TCID50/ml in 180 µl MucilAir™ medium placed onto the apical side of the cultures), noninfected control (Mock exposed to 200 µl of culture medium on the apical side), antiviral control with remdesivir (180 µl of 10 µM remdesivir dissolved in DMSO, and 20 µl of 4 x 105 TCID50/ml virus stock), control with DMSO alone, and control with Triton-X-100 (0.1% in MucilAir™ medium).
2.7. Titration of progeny virus
The titer of the apical washes (3, 24, 72, and 144 h post-infection) and the titer of the retrieved viral solutions from the mask material were determined using a plaque assay in a 24-well format in PFU per milliliter. Vero E6 cells were seeded in a 24-well plate at a density of 2.0 × 105 cells/ml 24 h before titration. After the cells were washed once with phosphate-buffered saline and the medium was changed, the cells were inoculated with apical washes containing viruses and serially diluted in a cell culture medium at 1:10 dilution. After 1 h of incubation, the inoculum was removed and subsequently overlaid with 1:1 mixture of 2.4% methylcellulose and 2× DMEM supplemented with 20% fetal bovine serum, 200 IU/ml penicillin, and 200 µg/ml streptomycin. After 48 h of incubation at 37°C, the cells were fixed in 4% (v/v) neutral-buffered formalin (Formafix AG, Hittnau, Switzerland) and stained with crystal violet. The number of wells displaying cytopathic effects was scored.
2.8 Biosafety and Biosecurity
All experiments with SARS-CoV-2 were performed in a BSL3 containment. A permit was issued by the responsible Swiss governmental authority for inactivation activities with SARS-CoV-2.
2.9. Genome copy quantification
Apical washes (20 µl) were used for viral RNA extraction with an E.Z.N.A. viral RNA kit (Omega Biotek, Norcross, USA). Viral RNA was quantified via quantitative RT-PCR (TaqMan Fast Advanced Master Mix, Thermo Fisher Scientific) by using 5 µl of viral RNA with Mastermix and the specific SARS-CoV-2 primers pWhSF-E-F21 (5ʹ-ACAGGTACGTTAATAGTTAATAGCGTACTTCT-3ʹ) and pWhSF-E-R22 (5ʹ-ACAATATTGCAGCAGTACGCACA-3ʹ) and one probe with FAM-MGB-Q530 reporter-quencher dyes. Four dilutions of a known concentration of SARS-CoV-2 and the controls for RNA extraction and RT-PCR were included, and the plates were run on QuantStudio™ 7 Flex PCR Detection System (Applied Biosystems, Thermo Fisher Scientific). Ct data were reported relative to the standard curve, corrected with the dilution factor, and presented as genome copy number per milliliter on the graphs.
2.10. Determination of epithelial integrity
Transepithelial electrical resistance (TEER) was measured to verify that all the selected inserts satisfy the internal quality control standards (TEER > 200 Ω·cm2) (Boda et al. 2018).
The integrity of the MucilAir™ epithelia was determined 24, 72, and 144 h post-infection by measuring the TEER of the tissues with an EVOM2™ voltohmmeter (World Precision Instruments UK, Stevenage, UK).
2.11. Data analysis
TEER values and virus replication performances between the test materials were statistically compared by using the R t-test function and two-sample Student’s t-test (two-tailed) with Welch modification to the degrees of freedom; the null hypothesis was that the true means of two groups were identical (Ripley 2001). These statistical comparisons were predefined based on scientific knowledge from literature. Data with p < 0.05 were considered significant, indicating that the null hypothesis should be rejected.