PMC:3463543 / 12181-17209 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":"Results\n\nIAV Viability in Model Media v. RH\nAs shown in Figure 1A and 1B, IAV viabilities in four model media containing various combinations of salts and proteins were highest at extremely high (∼100%) and low (\u003c50%) RH and minimal at medium (50 to 84%) RH. At ∼100% RH, viabilities ranged from 8% to 57%, namely, 36±15% in PBS, 7.8±0.27% in DMEM, 11±4.1% in PBS+FCS, and 57±11% in DMEM+FCS of input virus. At ∼84% RH, viabilities were much lower (∼1–5%) but were of the same order of magnitude in all four media.\nFigure 1 Relationship between RH and IAV viability in (A) media with mainly salts, (B) media with salts plus proteins, and (C) mucus. Error bars denote standard deviations. At RH between 50% and 84%, the trends in viability diverged dramatically, depending on whether the media contained proteins or not. The viability in PBS decreased with decreasing RH and reached a minimum at 50% RH. In DMEM, the trend was similar, but the minimum occurred at an RH of 60% (Figure 1A). In contrast, in PBS+FCS, viabilities did not change significantly over this range of RH, and in DMEM+FCS, viabilities increased slightly with decreasing RH (Figure 1B).\nAt RH below 50%, viabilities were higher in all types of media compared to those at medium RH, although viability in PBS alone was significantly lower than in the three other media, possibly due to the lack of proteins.\nWe repeated the experiment to confirm these results. The percent viabilities recovered varied slightly, but trends in viability versus RH were similar.\n\nIAV Viability in Mucus v. RH\nWe tested the changes in viability of IAV spiked into human mucus at RH levels ranging from ∼26 to ∼100%. As shown in Figure 1C, at ∼100% RH, the viability of IAV was only 1.7±0.26%, an order of magnitude lower than in model media. The finding that viability was highest at this extremely high RH was consistent with results in other media. At RH between ∼50% and 84%, viabilities increased dramatically with decreasing RH. Only 0.0027±0.0023% was recovered at 84% RH, compared to 1.5±1.4% recovered at 48% RH, a decrease of over two orders of magnitude. At RH below 48%, viabilities leveled off and remained around 1%. We repeated the experiment at four (27%, 60%, 84%, and 99%) of the six RHs, and the resulting viabilities followed the same trend as shown in Figure 1C. With spiking titers of 5.1±2.6×104 TCID50, no viable IAV was detected in 5 out of 9 samples at ∼84% RH.\n\nRelationship between Viral Decay and Salt Concentrations\nThe relationship between viral decay rates and salt concentrations in the droplets is shown in Figure 2. The four media used in this study and the mucus specimen contained multiple types of salts (Table 1). However, as NaCl was the major salt in all media and the mucus, we estimated the total concentrations of salts in the droplets at equilibrium in terms of equivalent amounts of NaCl; concentrations were estimated empirically [25]. The concentration of NaCl in a droplet is related to RH; however, as indicated by the polynomial function in Eq. 3, the correlation is not simply linear. The viral decay rate in PBS droplets increases linearly with NaCl concentration in the range of 25–510 g L−1, corresponding to 99–50% RH, respectively (R2 = 0.97). Concentrations are lower at high RH because there is less evaporation and vice versa. Likewise, the viral decay rate in DMEM droplets also increases linearly with NaCl concentration up to 420 g L−1, corresponding to 60% RH (R2 = 0.98). In contrast, in media containing FCS, viral decay rates remain constant regardless of NaCl concentration.\nFigure 2 Viral decay over 3 h versus NaCl concentration in droplets consisting of four types of media.\n\nEfflorescence\nAt RH below ∼50%, the relationship between IAV decay rates and NaCl concentrations is no longer valid. NaCl reaches its solubility in a droplet (310 g L−1) at 75% RH, and evaporation produces a supersaturated solution at RH\u003c75%. The NaCl concentration can rise as high as 580 g L−1 before the droplet crystallizes [25], [26]. The critical RH at which a droplet crystallizes is termed the efflorescence RH (ERH), and it depends on the composition and size of the droplet [27].\nWe observed the droplets of each medium under a microscope immediately after incubation at different RHs and found that no crystals formed at RH\u003e60%, while droplets of all media crystallized at an RH just below 50% (Figure 3). Crystallization occurred within ∼10 min. Crystallization in DMEM, possibly at a lower NaCl concentration (corresponding to higher RH) than in PBS, due to a different ERH of the media, could explain the lower decay rate in DMEM at a concentration of 510 g L−1 (50% RH). Knowing the ERH of each media would lend further support to this idea, but the ERH of a solution is difficult to calculate and can only be measured accurately through specialized laboratory experiments [27].\nFigure 3 Crystals of the four media: (A) PBS, (B) PBS+FCS, (C) DMEM, (D) DMEM+FCS. 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