PMC:7723248 / 7798-15071 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7723248","sourcedb":"PMC","sourceid":"7723248","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7723248","text":"Materials and methods\n\nVirus stock\nBacteriophage MS2 (ATCC 15597-B1), a model for non-enveloped RNA viruses that is widely used in environmental engineering studies [33–35], was propagated as previously described [12]. Briefly, E. coli (ATCC 15597) was inoculated in Miller’s Luria Broth (LB) medium (Fisher Scientific) and incubated overnight at 37°C. Fifty microliters of MS2 stock, 450 μL of E. coli liquid culture, and 4.5 mL of LB soft agar were mixed and overlaid on LB agar plates. Plates were incubated at 37°C for 24 h before the top layer of soft agar was collected in LB medium. The mixture of soft agar and LB medium was shaken at 100 rpm at 37°C for 2 h. The mixture was then centrifuged at 5000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm cellulose acetate membrane to remove cells and debris. The filtrate was collected as virus stock and stored at 4°C.\nBacteriophage Φ6 (kindly provided by Dr. Paul Turner at Yale University), a model for enveloped RNA viruses including influenza virus [4, 35–37], was propagated using the same method described above, except that the bacterial host was Pseudomonas syringae, which grows at 25°C. Determined by plaque assay, the concentrations of virus stocks were 1010−1011 plaque-forming units (PFU)/mL.\n\nPreparation of virus suspension\nFor testing the effect of different components of media on virus viability, solutions containing various amounts of salt, protein, and surfactant were prepared. Specifically, a 100 g/L NaCl stock solution was prepared by adding 100 g of sodium chloride (Fisher Scientific) to ultrapure water (Barnstead Nanopure; Thermo) to a final volume of 1 L. Aliquots of the solution were then diluted with ultrapure water to produce working solutions with the concentrations shown in Table 1. Stock solutions of bovine serum albumin (BSA) (Sigma), a protein derived from cows, and sodium dodecyl sulfate (SDS) (Sigma), an anionic surfactant used in many cleaning and hygiene products, were prepared and diluted similarly as the NaCl stock solution.\nTable 1 Chemicals used to make solutions and their concentrations in virus suspensions.\nComponent Chemical(s) used Stock solution concentration Concentration/pH tested Relevance\nSalt Sodium chloride 100 g/L 0, 1, 35 g/L Salinity of seawater: 35 g/L\nSalinity of surface water: 0.01 to few g/L [38]\nLung fluid: ~10 g/L [39]\nProtein Bovine serum albumin 100 mg/mL 0, 100, 1000 μg/mL Respiratory fluid: 30–8500 μg/mL [25, 40–42]\nSurfactant Sodium dodecyl sulfate 100 μg/mL 0, 1, 10 μg/mL Lung surfactant: up to 1000 μg/mL [19, 43]\nSurface water: less than 1 μg/mL [20]\npH Hydrochloric acid, sodium hydroxide NA 4.0, 7.0, 10.0 Ambient aerosol: 0–4 [44, 45]\nInorganic aerosol: basic [46]\nHuman respiratory fluid: pH-neutral [47]\nNA indicates no stock solution with different pH values was prepared. Virus suspensions at the targeted concentrations of salt, protein, and surfactant were prepared right before experiments were conducted. Virus stock and the working solutions were mixed at a ratio of 1:100. Specifically, 50 μL of virus stock was diluted with 4.95 mL of the working solution of interest in 15 mL centrifuge tubes and vortexed for 30 seconds. Virus stock and ultrapure water, which had an initial pH of 5.5, were mixed at the same ratio. The pH of the mixture was adjusted to 4.0, 7.0, or 10.0 with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide and was measured using a pH meter (Orion Versa Star; Thermo). To avoid introducing excessive ions to the mixture, no pH buffer was added after the pH was adjusted to the target value in the mixture. The amount of ions introduced to the solutions to adjust the pH, shown in Table 1, was much lower than that used to make the 1 g/L NaCl working solution. Thus, the change in ionic strength due to pH adjustment should have a negligible effect on virus inactivation.\n\nViability of viruses in droplets\nThe viability of viruses in droplets was studied in an environmental chamber (5518; Electro-Tech Systems) at room temperature (22 ± 1°C). For each virus suspension, droplets were exposed at low, intermediate, and high RH levels of 20%, 50%, and 80%, respectively. The targeted RH inside the environmental chamber was achieved by vaporizing ultrapure water with a humidifier or passing air through a desiccator. Fifteen minutes after the RH reached equilibrium, ten separate 1-μL droplets of virus suspension were spotted on a 6-well, polystyrene cell culture plate (SIAL0516; Sigma) with a 0.1-10-μL pipette. Droplets were incubated for 1 h, after which viruses were collected in 500 μL of LB medium by pipetting up and down several times. Samples were stored at -80°C immediately after collection until they were quantified by plaque assay. Control samples containing 10 μL of virus suspension in a sealed 1.5-mL microcentrifuge tube were incubated inside the environmental chamber during each experiment and collected in 500 μL of culture medium after 1 h.\n\nPlaque assay and relative viability\nVirus samples were quantified by plaque assay as described previously [12]. Briefly, 10-fold serial dilutions of the collected samples were prepared. Fifty microliters of the serial dilutions, 450 μL of liquid culture of bacterial host, and 4.5 mL of soft agar were mixed and poured over agar plates. Plates were incubated at the bacterial host’s growth temperature for 24 h. The number of plaques on plates was counted, and the virus concentration in the samples were calculated as shown in Eq 1.\nV i r a l t i t e r ( P F U / m L ) = # o f p l a q u e s d i l u t i o n f a c t o r * v o l u m e o f v i r u s s u s p e n s i o n a d d e d t o t h e p l a t e (1) The change in infectious viral concentration after 1 h of exposure was expressed as relative viability. Relative viability was calculated as the ratio of the post-exposure viral concentration, Cpost-exposure, to the pre-exposure concentration, Cpre-exposure, as shown in Eq 2.\nR e l a t i v e V i a b i l i t y = C p o s t − e x p o s u r e C p r e − e x p o s u r e (2)\n\nDroplet evaporation rate\nThe droplet evaporation rate was determined as described previously [13]. Briefly, a microbalance (MSE3.6P; Sartorius) was placed inside an environmental chamber to weigh ten 1-μL droplets, which were spotted on a microscope cover glass (12-545-M; Fisher Scientific), over 1 h. Droplet mass was recorded at 1-min intervals. Droplet evaporation rate was calculated as shown in Eq 3. Dropletevaporationrate=dmdt=mtn−mtn−1(3) Where m is the mass of droplets (mg), and tn is n min after droplets were spotted.\n\nStatistical analysis\nAll experiments were performed in triplicate. The relative viability of viruses was expressed as mean ± standard deviation. One-way ANOVA and a post-hoc analysis with Tukey’s HSD test were performed to determine significant differences (P \u003c 0.05) in the relative viability of bacteriophages among different levels of media composition and among RH levels, respectively. Two-way ANOVA was performed to determine the interaction effect between media composition and RH. One-way ANOVA was performed to determine significant differences (P\u003c0.05) in the evaporation rate of droplets with different initial solute concentrations.","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"sec","span":{"begin":23,"end":1277}},{"label":"title","span":{"begin":23,"end":34}},{"label":"p","span":{"begin":35,"end":890}},{"label":"p","span":{"begin":891,"end":1277}},{"label":"sec","span":{"begin":1279,"end":3890}},{"label":"title","span":{"begin":1279,"end":1310}},{"label":"p","span":{"begin":1311,"end":2048}},{"label":"table-wrap","span":{"begin":2049,"end":2862}},{"label":"label","span":{"begin":2049,"end":2056}},{"label":"caption","span":{"begin":2058,"end":2137}},{"label":"title","span":{"begin":2058,"end":2137}},{"label":"table","span":{"begin":2138,"end":2792}},{"label":"tr","span":{"begin":2138,"end":2231}},{"label":"th","span":{"begin":2138,"end":2147}},{"label":"th","span":{"begin":2149,"end":2165}},{"label":"th","span":{"begin":2167,"end":2195}},{"label":"th","span":{"begin":2197,"end":2220}},{"label":"th","span":{"begin":2222,"end":2231}},{"label":"tr","span":{"begin":2232,"end":2306}},{"label":"td","span":{"begin":2232,"end":2236}},{"label":"td","span":{"begin":2238,"end":2253}},{"label":"td","span":{"begin":2255,"end":2262}},{"label":"td","span":{"begin":2264,"end":2276}},{"label":"td","span":{"begin":2278,"end":2306}},{"label":"tr","span":{"begin":2307,"end":2354}},{"label":"td","span":{"begin":2307,"end":2354}},{"label":"tr","span":{"begin":2355,"end":2379}},{"label":"td","span":{"begin":2355,"end":2379}},{"label":"tr","span":{"begin":2380,"end":2486}},{"label":"td","span":{"begin":2380,"end":2387}},{"label":"td","span":{"begin":2389,"end":2409}},{"label":"td","span":{"begin":2411,"end":2420}},{"label":"td","span":{"begin":2422,"end":2440}},{"label":"td","span":{"begin":2442,"end":2486}},{"label":"tr","span":{"begin":2487,"end":2592}},{"label":"td","span":{"begin":2487,"end":2497}},{"label":"td","span":{"begin":2499,"end":2521}},{"label":"td","span":{"begin":2523,"end":2532}},{"label":"td","span":{"begin":2534,"end":2548}},{"label":"td","span":{"begin":2550,"end":2592}},{"label":"tr","span":{"begin":2593,"end":2630}},{"label":"td","span":{"begin":2593,"end":2630}},{"label":"tr","span":{"begin":2631,"end":2721}},{"label":"td","span":{"begin":2631,"end":2633}},{"label":"td","span":{"begin":2635,"end":2670}},{"label":"td","span":{"begin":2672,"end":2674}},{"label":"td","span":{"begin":2676,"end":2690}},{"label":"td","span":{"begin":2692,"end":2721}},{"label":"tr","span":{"begin":2722,"end":2751}},{"label":"td","span":{"begin":2722,"end":2751}},{"label":"tr","span":{"begin":2752,"end":2792}},{"label":"td","span":{"begin":2752,"end":2792}},{"label":"table-wrap-foot","span":{"begin":2793,"end":2862}},{"label":"footnote","span":{"begin":2793,"end":2862}},{"label":"p","span":{"begin":2793,"end":2862}},{"label":"p","span":{"begin":2863,"end":3890}},{"label":"sec","span":{"begin":3892,"end":4983}},{"label":"title","span":{"begin":3892,"end":3924}},{"label":"p","span":{"begin":3925,"end":4983}},{"label":"sec","span":{"begin":4985,"end":6094}},{"label":"title","span":{"begin":4985,"end":5020}},{"label":"p","span":{"begin":5021,"end":5518}},{"label":"label","span":{"begin":5706,"end":5709}},{"label":"p","span":{"begin":5712,"end":5988}},{"label":"label","span":{"begin":6091,"end":6094}},{"label":"sec","span":{"begin":6096,"end":6627}},{"label":"title","span":{"begin":6096,"end":6120}},{"label":"p","span":{"begin":6121,"end":6627}},{"label":"label","span":{"begin":6540,"end":6543}},{"label":"title","span":{"begin":6629,"end":6649}}],"tracks":[]}