PMC:3463543 / 5271-12179 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3463543","sourcedb":"PMC","sourceid":"3463543","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3463543","text":"Materials and Methods\n\nEthics Statement\nCollection and use of human mucus was approved by the Institutional Review Board at Virginia Tech. The ethics committee did not require consent to be obtained for this protocol.\n\nCells and Virus\nMadin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS). The stock of the IAV A/PR/8/34 (H1N1) was prepared with a plaque purified strain [22] in 10-day-old specific pathogen-free embryonated hens’ eggs. Virus stocks were stored as aliquots of allantoic fluid from infected embryos at −80°C until use. The titer of the virus stock was determined to be 1.78×108 median tissue culture infective dose (TCID50) mL−1. All virus titration experiments were performed in MDCK cells.\n\nMucus Specimen\nA mucus specimen was collected from an infant 1 month of age in March 2011. The mucus specimen was allowed to desiccate in a vial and was reconstructed by dissolution in MilliQ water (5% w/v). To preserve the mucus structure, we first attempted to sterilize the mucus specimen by passing it through a 0.45-µm pore size filter. The specimen was not filterable, indicating its large molecular size. Thus, the reconstructed mucus was sterilized by heating at 65°C for 30 min and was checked for potential IAV contamination by TCID50 assay. No IAV was detected, and the specimen was stored at 4°C until use.\n\nControl of RH in a Desiccator\nTwo Petri dishes filled with 20 mL of salt solution (either potassium acetate, potassium carbonate, cobalt (II) chloride, or potassium chloride) or double distilled water were placed in a desiccator [23], [24]. Air circulation inside the desiccator was enhanced by a fan to accelerate the establishment of equilibrium RH between the salt solution and the air inside the desiccator. Before each experiment began, the desiccator was conditioned to the desired RH. The desiccator had to be opened briefly to insert the samples, and the system reestablished equilibrium within ∼10 min. The actual RH and temperature inside the desiccator were recorded at 1-min intervals during sampling with a temperature/humidity logger (OM-73, Omega Engineering, Inc., USA). RHs tested in model media included 17.5±1.0%, 27.6±0.9%, 42.0±0.8%, 44.1±1.2%, 50.2±0.7%, 59.5±1.1%, 76.1±0.7%, 84.1±0.8%, and 98.9±0.4%. RHs tested in mucus included 26.7±0.8%, 40.3±1.1%, 48.0±1.2%, 52.0±0.9%, 60.8±1.1%, 72.2±1.2%, 83.9±0.4%, and 99.1±0.4%.\n\nExposure of IAV in Droplets to Various RH\nWe exposed IAV in droplets to specific RHs in the range of 17% to ∼100% at room temperature (20–24°C). The droplets consisted of four different types of media (Table 1), chosen to isolate the effects of salts and/or proteins on viability, or mucus. PBS and DMEM were used as surrogates of media that contain inorganic salts at physiological levels but no or negligible amounts of proteins. The third and fourth types of media were PBS and DMEM supplemented with 5% FCS, a source of proteins.\nTable 1 Media used in studies of influenza A virus viability versus RH.\nStudy Media Salt content Protein content Other components Reference\nHemmes et al. 1960 1 part allantoic fluid and one part2% Difco peptone ∼2.3 g L−1 (0.4 g L−1 K+, 0.9 g L−1 Na+,0.9 g L−1 Cl−)a 10 g L−1 peptone -b [8], [43]\nHarper 1961 Allantoic fluid diluted 1∶8 or 1∶10 incasein McIlvaine’s buffer (pH 7.2) ∼2.2 g L−1 (mainly Na2HPO4) a ∼1.9 g L−1 mainly casein -b [13], [43], [44]\nShechmeister 1950 Allantoic fluid in 0.1 M Sorensen’sphosphate buffer (pH 7.1) 19.6 g L−1 (8.09 g L−1 NaH2PO4, 9.51 gL−1 Na2HPO4) ∼1 g L−1 in allantoicfluid -b [15], [43]\nSchaffer et al. 1976 MEM 9.88 g L−1 (6.8 g L−1 NaCl, 2.2 g L−1NaHCO3, and others) 0 Amino acids, vitamins,glucose, others [14], [43], [45]\nMEM+0.1% BSA 9.88 g L−1 1 g L−1 Same as above\nAllantoic fluid ∼4.7 g L−1 (0.8 g L−1 K+, 1.8 g L−1 Na+,1.8 g L−1 Cl−)a ∼1 g L−1\nThis work PBS (pH 7.2) 9.55 g L−1 (8 g L−1 NaCl, 0.2 g L−1 KCl,1.15 g L−1 Na2HPO4, 0.2 g L−1 KH2PO4) 0 None\nPBS+5% fetal calf serum (FCS) Same as above ∼3.5 g L−1 Other componentsfrom FCS\nDulbecco's modified Eaglemedium (DMEM) 10.92 g L−1 (6.4 g L−1 NaCl, 3.7 g L−1NaHCO3) 0.42 g L−1 Amino acids, vitamins,glucose, others\nDMEM+5%FCS Same as above ∼3.9 g L−1 Same as above\na Estimated;\nb Detailed composition of allantoic fluid is unknown. Ten µL of stock PR/8 IAV was added into 90 µL of either PBS, PBS+5% FCS, DMEM, DMEM+5% FCS, or mucus to produce spiking solutions. The spiking solutions were distributed onto a 12-well cell culture plate, 1 µL per droplet, 10 droplets per well, and 3 replicates (i.e., 3 wells) for each medium. The plate was immediately placed into the desiccator and was incubated at a specific RH at room temperature for 3 h for model media and 2 h for mucus. At the end of the period, the virus in each well was collected with 1 mL of DMEM supplemented with 1 µg mL−1 TPCK trypsin (collection medium), by pipetting the medium ∼10 times to rinse the virus from the well. The spiking solutions were stored on ice during the incubation period, and 10 µL of each spiking solution was supplemented with 990 µL of collection medium for use as a control. Samples and the corresponding controls were titrated at the same time by TCID50 assay either immediately after collection or were stored at −80°C until testing.\n\nObservation of Transformation of Droplets under Varying RH Levels\nDroplets of PBS, PBS+5% FCS, DMEM, or DMEM+5% FCS (all without addition of IAV) in 1-µL volumes were distributed on a 12-well cell culture plate and incubated under different RH levels as described above. Plates were taken from the incubator and observed immediately under an inverse light microscope (100X). Photos were taken from a camera connected to the microscope. To determine the time needed for a droplet of 1 µL to dry out, droplets of PBS, PBS+5% FCS, DMEM, or DMEM+5% FCS were distributed on a glass slide, left under the microscope and observed continuously until the droplet crystallized. Ambient RH was 49.4±0.2% in the microscope room.\n\nCalculation of Viral Decay\nPercentage viability was calculated as(1) where Nt and N0 are, respectively, the final and initial titers of virus. The log decay rate was calculated as(2) Nt was set to 1 for negative samples to avoid dividing by 0.\n\nEstimation of Solute Concentration in Equilibrium\nThe molality of NaCl in the resulting droplet at a specific RH was calculated using Microsoft Excel’s solver tool according to an empirical relationship [25]:(3) where m is the molality of NaCl (mol (kg water)−1). The concentration of NaCl was then calculated based on m:(4) where Cs is the concentration of solute in equilibrium; Ms is the molecular weight of NaCl (58.44 g mol−1); vs is the molar volume of NaCl (2.7×10−2 L mol−1); and the number 1 in the denominator represents the volume of 1 kg of water, i.e., 1 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