Bare and salt-coated filter samples preparation

The commercial surgical masks had a three-ply structure. The middle layer is the filter media, whereas the inner and outer layers provide support and protect the filter against wear and tear. The metal nose clips and elastic ear loops were removed and circular samples (radius: 3 cm) were cut from the masks. The PP filters (middle layer) were isolated by removing the inner and outer protective layers (bare filters, Filter bare ). The coating solution was prepared by dissolving sodium chloride (NaCl; Sigma Aldrich, St. Louis, MO) in filtered DI water (0.22 μm pore size; Corning, Tewksbury, MA) under stirring at 400 rpm and 90 °C, followed by the addition of Tween 20 (Fisher Scientific) to a final concentration of 29.03 w/v% of NaCl and 1 v/v% of Tween 20. To obtain the salt-coated filters, the mask bare PP filters were pre-wet to contain approximately 600 μL of coating solution by incubating overnight at room temperature. Any remaining dry areas were removed by applying gentle strokes with tweezers to the filters while immersed in the coating solution. Subsequently, the filters were deposited in the desired volume of coating solution (0, 100, 300, 600, 900 and 1200 μL, of which corresponding membranes are abbreviated as Filter wet , Filter wet+100μL , Filter wet+300μL , Filter wet+600μL , Filter wet+900μL , and Filter wet+1200μL , respectively) on petri dishes (60 × 15 mm; Fisher Scientific) to control the amount of NaCl per unit area and dried in an oven (Isotemp Incubator, Fisher Scientific) at 37 °C for 1 day.

Influenza virus preparation

Influenza viruses A/California/04/2009 (CA/09, H1N1), A/Puerto Rico/8/34 (PR/34, H1N1) and A/Vietnam/1203/2004 (VN/04, H5N1) were grown in 10-day old embryonated hen eggs, in which H5N1 virus was derived by reverse genetics from HPAI A/Vietnam/1203/200436. Influenza viruses were purified from allantoic fluid using discontinuous sucrose gradient (15%, 30% and 60%) layers following the previously reported procedure37.

Aerosols exposure to filters

For experiments involving aerosols exposure, an aerosol chamber (L × W × H = 145 × 145 × 150 mm; Emka Inc., Middletown, PA) was used (Fig. S11). It has a connection to the vacuum line and a circular aperture in the top wall (diameter: 22 mm) to exactly accommodate the cylindrical part (diameter: 20 mm, height; 20 mm) of the nebulizer unit that is below the aerosol generator (Aeroneb Lab Nebulizer System; Aerogen, Galway, Ireland). Bleach was used as trap between the chamber and the vacuum pump (Welch 2522C-10, 22 L/min; Niles, IL). The filters were placed on top of the chamber aperture and the nebulizer unit was inserted, ensuring the tight seal of the filters against the side of the aperture. 5 μL of virus stock were added to the nebulizer unit, aerosols (VMD 2.5–4 μm from manufacturer specifications) were generated for 30 sec and subsequently the desired vacuum level (3, 10 or 17 kPa) was applied, by manual control, three times in 1 sec cycles. Notably, in the case of bare filters, pressure was only applied for filtration efficiency tests.

For all assays and analysis, suspensions of the filters were prepared as follows, unless otherwise indicated. To reconstitute virus adsorbed onto filters, virus-laden filters were immersed in 400 μL of sterilized DI water for about 5 min, and then removed after vortexing from the suspension. The virus suspension was centrifuged at 19,800 g and 4 °C for 10 min (Centrifuge 5810 R, Eppendorf, Hauppauge, NY), followed by resuspension of pellets in 70 μL of DI water to eliminate any interference from materials in supernatant during assays.

Filtration efficiency tests

The filters were exposed to the virus aerosols at 3, 10 and 17 kPa and suspensions of the filters were obtained, as described above. The filtration efficiency was calculated as the ratio of the amount of virus (i.e., total proteins measured from the virus) reconstituted from the filter to that from the virus in the exposure aerosols. The concentration of virus in aerosols was determined by generating viral aerosols into a 15 mL centrifuge tube, containing 1 mL of DI water. After vortexing, virus concentrations (i.e., total protein concentration) were measured with bicinchoninic acid assay (BCA protein assay kit; Thermo Fischer scientific, Waltham, IL) with bovine serum albumin as a standard. In the case of virus reconstituted from salt-coated filters, virus-laden filter suspension was replaced with DI water prior to BCA assay.

In vivo infection tests

Lethal infectivity of influenza viruses (CA/09 H1N1) was examined in 8 week old female inbred BALB/c mice (Nara Biotech; Seoul, Korea) by using the intranasal route. For bare and salt-coated filters, 12 mice per group were infected with individual penetration dosage of influenza virus through each filter. The penetration dosage of the virus through the filters (Filter bare , Filter wet , Filter wet+600μL , and Filter wet+1200μL ) was calculated from the filtration efficiency at 10 kPa (near breathing pressure) using the relationship: penetration dosage = virus dosage in lethal aerosol × penetration efficiency (%)/100, where penetration efficiency (%) = 100 − filtration efficiency (%). To examine the effects of the aerosolization process on the viral infectivity change, two mice groups were infected with a lethal dose of virus before and after aerosol formation, which served as negative control groups. Body weight changes and survival rate of mice were monitored daily for 15 days. Mice with body weight loss greater than 25% were euthanized. All animal protocols were approved by the Kyung Hee University (KHU) Institutional Animal Care and Use Committee (IACUC). All animal experiments and husbandry involved in this work were conducted under the approved protocols and guidelines of KHU IACUC. KHU IACUC operates under National Veterinary Research and Quarantine Service (NVRQS), and animal welfare law and regulations of the WOAH-OIE (World organization for animal health).

To test strain-dependent lethal infection behavior, mice (12 per group) were infected with the penetrated dosage of viral aerosols (PR/34 H1N1 and VN/04 H5N1 viruses) through Filter wet+600μL at 10 kPa. Time-dependent body weight change was monitored in the same manner described above.

Lung viral titer and lung inflammatory cytokine assays after infection

On day 4 after infection 6 mice of each group were sacrificed for the collection of lung samples. Lung virus titers were measured on six-well plates containing confluent MDCK cell monolayers. Inflammatory cytokines (IFN-γ) were determined using BD OptEIA mouse IFN-γ ELISA kit (BD Biosciences, San Jose, CA) following the manufacturer’s procedure.

Test of viral infectivity change on filters

To investigate the effects of salt-coating on viral infectivity loss, lethal influenza aerosols were exposed to four different types of filters (Filter bare , Filter wet , Filter wet+600μL , and Filter wet+1200μL ). Since Filter bare exhibited almost complete penetration upon pressure application, aerosols were exposed to the bare filter in the absence of pressure and samples were carefully handled to prevent mechanical agitation. To measure time-dependent stability change of virus, virus-laden filters were incubated at ambient conditions for 0, 5, 15, and 60 min after aerosol exposure, and suspended in DI water to reconstitute virus at each time point. In vitro stability of virus was characterized by measuring hemagglutinin activity (HA) and virus titers at the same concentration as lethal dose30. The conformational stability of antigenic proteins was characterized by measuring intrinsic fluorescence using 0.1 mg/mL of virus suspension38. To investigate morphological change of virus, lipid stability of viral wall was characterized by nile red fluorescence (Sigma Aldrich), a fluorescent lipid stain, following manufacturer’s protocol39. A decrease in fluorescence intensity can be used to examine the level of disintegration of the virus. Both intrinsic and nile red fluorescence were measured by using a fluorimeter (LB 50B; PerkinElmer, Waltham, MA). Intensity changes of fluorescent spectra were compared relative to those of a control from virus stock.

To test infectivity difference observed from in vitro findings, in vivo study was performed for the virus reconstituted from the filters (Filter bare , Filter wet , Filter wet+600μL , and Filter wet+1200μL ) after incubation for 60 min at RT (aerosol exposure at 10 kPa, except for Filter bare ). 12 mice per group were infected with a lethal dose of virus collected from each type of filter. Body weight change and lung virus titers were measured as described above.

Effects of environmental conditions on the performance of salt-coated filter

Salt-coated filters (Filter wet , Filter wet+600μL , and Filter wet+1200μL ) were stored at 37 °C, 70% RH in an incubator (Maru Max; Rcom, Gyeonggi-do, South Korea) for 15 days. Every day, the filters were collected and incubated at ambient conditions for 5 min. At 1-day incubation, filtration efficiency was measured at 10 kPa from Filter wet+600μL , followed by in vivo infection test. Lethal infectivity between two different filter groups (before and after incubation at 37 °C, 70% RH) was compared by measuring body weight change and survival rate of mice after exposure to lethal CA/09 H1N1 aerosols. XRD analysis was performed to salt-coated filters incubated for 1 and 15 days, and SEM/EDX mapping analysis for 15-day incubated samples.

Contact angle measurements and imaging of aerosols

The bare and salt-coated filters were fixed with carbon tape (Ted Pella, Inc., Redding, CA) to a metal, flat substrate and 3 μL of DI water were added on the surface of the filters. The contact angles were measured from images collected with an optical microscope (10× lens, Motic SMZ-140; Motic, Richmond, Canada) at RT. Images of aerosols on filter fibers were obtained using a dispersive Raman microscope (Nicolet Almega XR; Fisher Scientific).

Aerosol drying time on filters

The bare and salt-coated filters were fixed with carbon tape to a metal, flat substrate and exposed to aerosols generated from 5 μL of Sulforhodamine B Dye solution (1 mM, Sigma-Aldrich). Aerosol drying time was determined with timer by observation with optical microscope.

Electron microscopy analysis

For virus stability tests, bare and salt-coated filters were exposed to CA/09 H1N1 aerosols and, after 5 and 60 min incubation, virus was recovered by suspension of the filters, as described above. To study the effects of the coating formulation during aerosol drying independently from crystal growth, bare and salt-coated filters were immersed in DI water and removed after 60 min. Subsequently, virus was incubated in the obtained suspension for 60 min. Additionally, the virus suspension was centrifuged at 19,800 g and 4 °C for 10 min to collect the samples and suspend them in DI water. For TEM analysis (200 kV, JEOL JEM 2100; JEOL, Peabody, MA), samples were deposited on copper grid (Electron Microscopy Sciences, Hatfield, PA) and negatively stained with solution comprised of phosphotungstic acid hydrate (1.5 w/v%, pH = 7.0; Sigma-Aldrich, Oakville, Canada).

To identify the morphology of salt-coated filters and recrystallized salts, SEM/EDX analysis was performed for bare and salt-coated filters after coating with 10 nm thick gold layer. Scanning electron microscopy analysis (Hitachi S-3000N; Hitachi, Toronto, Canada) was operated in secondary electron mode at 20 kV and EDX analysis was obtained with EDX detector (Oxford Instruments, Concord, MA).

XRD analysis

To confirm the formation of crystalline NaCl coating during drying process and its stability during storage at 37 °C and 70% RH, XRD analysis (BRU-1098; Bruker, Billerica, MA) was performed at different coating conditions. Filters (1 × 1 cm) were mounted on a slide glass for XRD analysis (θ–2θ mode) using a CuKα radiation.

Statistical analysis

To compare multiple conditions, Student’s t-test, One-way analysis of variance (ANOVA), and general linear model were used (Minitab release 14; Minitab, State College, PA). P value of less than 0.05 was considered to be significant.