Exposure of IAV in Droplets to Various RH
We 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.
Table 1  Media used in studies of influenza A virus viability versus RH.
Study  Media  Salt content  Protein content  Other components  Reference
Hemmes 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]
Harper 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]
Shechmeister 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]
Schaffer 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]
MEM+0.1% BSA  9.88 g L−1  1 g L−1  Same as above
Allantoic 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
This 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
PBS+5% fetal calf serum (FCS)  Same as above  ∼3.5 g L−1  Other componentsfrom FCS
Dulbecco'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
DMEM+5%FCS  Same as above  ∼3.9 g L−1  Same as above
a  Estimated;
b  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.