PMC:3463543 / 17211-29562 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":"Discussion\n\nHypothesis to Explain the Relationship between RH and Viability in Laboratory Studies\nWe postulate that there exist three regimes governing the viability of IAV in droplets, defined by ambient RH and shown in Figure 4: (1) physiological conditions (∼99 to 100% RH), where solute concentrations remain at levels harmless to IAV and viability is maintained, (2) concentrated conditions (∼50 to ∼99% RH), where evaporation leads to elevated salt concentrations that may be harmful to the virus, and (3) dry conditions (\u003c50% RH), where solutes crystallize, all water is lost, and IAV viability is maintained.\nFigure 4 Hypothesized relationship between RH and IAV viability in (A) droplets containing salts only and (B) droplets containing salts plus proteins. In a saline droplet, the elevated salt concentrations that result from evaporation are likely to be toxic to the virus, but such deleterious effects would be eliminated when the solution crystallizes. Therefore, the minimum viability would be expected at an RH just above the ERH of the salts contained in the droplet, when water is still present and solute concentrations are maximal. The relationship between RH and IAV viability in a saline droplet would thus be similar to that shown in Figure 4A. However, the presence of proteins in the droplet may alter this relationship. It is possible that the interaction between proteins, salts, and the virus mitigates the adverse effects of salts under concentrated conditions. Therefore, the virus would maintain high viability under physiological and dry conditions and moderate viability under concentrated conditions in a droplet composed of both salts and proteins (Figure 4B). In fact, viability may increase with decreasing RH, as we found in DMEM+FCS and mucus, possibly due to protection provided by proteins at elevated concentrations [28], [29].\nA study on Langat virus supports our hypothesis. Benbough [30] reported similar V-shaped curves of viability versus RH for Langat virus in aerosols composed of salt solutions. Of four RHs tested (i.e., ∼25%, 50%, 70%, and 95%), the minimum viabilities were ∼1% in NaCl and ∼10% in KCl, both at ∼50% RH, and zero in LiCl at RH\u003c50%. Thus, the minimum viability of Langat virus in aerosols composed of an NaCl solution occurred at an RH close to the ERH of NaCl (i.e., 43±3%) [27]. KCl has an ERH of 59% [27], and this RH was not tested, so it is unknown whether the virus’ viability would have been lower at this RH. The ERH of LiCl, if it exists, is outside the range of RHs tested; its deliquescence RH is ∼11% [24].\n\nExplanation for Discordant Findings in Literature\nWhen reviewing the four aforementioned studies (H\u0026H versus S\u0026S), we noticed that the media used to produce airborne droplets containing IAV differed. All the media contained some salts; however, those in H\u0026H contained substantially more proteins than did those in S\u0026S (Table 1). We thus hypothesize that the conflicting results between S\u0026S and H\u0026H were due to the varying protein content of the media used in these studies. Our results with model media confirm this hypothesis: the trends in viability v. RH in media with mainly salts (S\u0026S and this study) resemble the illustration in Figure 4A, while those in media with both salts and proteins (H\u0026H and this study) resemble Figure 4B.\nWe showed that viral decay is correlated with the concentration of salts in saline droplets, which is controlled by RH through evaporation (Figure 2). Previous studies based on experiments in bulk salt solutions at concentrations up to saturation failed to detect such a correlation [31]. The supersaturated conditions to which the virus is exposed in aerosols cannot be achieved in bulk media, and so the phenomenon observed here can only be demonstrated through methods that enable supersaturation. The combination of highly elevated salt concentrations at medium RH and salt crystallization at the ERH may explain the trends observed in the two media containing mainly salts (i.e., PBS and DMEM) and in S\u0026S. The RH corresponding to the maximal decay rate may differ between media, possibly due to different ERHs for media of different compositions [27]. The minimum viabilities occurred between 40–70% RH, mostly around 50% in Schaffer et al. [14] and 58–60% in Shechmeister. [15] For comparison, we found minimum viabilities at 50% in PBS and 60% in DMEM.\nAs did H\u0026H, we found that proteins had a “protective” effect for IAV under concentrated conditions at medium RH, although the reasons for it are still unknown. One possibility is that proteins in aerosol droplets could become enriched around viruses due to mutual hydrophobicity and provide some protection against concentrated salts. Although this hypothesis is highly speculative, it is supported by the fact that mucin glycoproteins usually serve as a barricade against potential pathogens including viruses and bacteria by specific or non-specific binding [28], [29].\n\nRelationship in Mucus\nRespiratory tract mucus is a complicated combination of water (∼95%), inorganic salts (∼1%), and various macromolecular organic compounds including glycoproteins (i.e., mucins) and lipids [20], [21], [32]. It has been shown to be protective for IAV at low RH [33], [34]. However, the relationship between viability of IAV in mucus and RH is still not completely clear.\nWe initially conducted experiments with mucin extracts from porcine stomach (Type II, Sigma-Aldrich). Results indicated that IAV infection was blocked, probably by the bound sialic acid on the mucin. Similarly, glycans on human mucus could prevent IAV attachment to the cell in the TCID50 assay. However, such an effect was not significant according to our experiment. The titer of IAV spiked in the human mucus specimen was 5.1±2.6×107 TCID50 ml−1 after being kept on ice for 2 h, with a spiking titer of 1.78×108 TCID50 ml−1. In addition, reporting results in mucus samples as the ratio of the recovered viability after incubation at a specific RH over the initial viability, both tested in mucus, should control for any potential blocking effect due to glycans on mucus. Therefore, results reported here reflect the effect of RH.\nIn accordance with the literature [33], [34], we found higher viabilities at low RHs (\u003c50%). The finding that IAV survived best at ∼100% RH, a condition which has not been examined previously, helps complete the understanding of the response of the virus to varying RH. The relationship in mucus bears some similarity to that in media with salts plus proteins, particularly DMEM+FCS (i.e., higher viabilities at ∼100% RH or RH\u003c50% and much lower ones at medium RH ranging from ∼50% to 84%). However, viability was much more sensitive to RH in mucus than in synthetic model media. A small change in RH from 48% to 52% reduced the viability 10-fold, and viability at ∼84% RH was \u003e500 times lower than at 48% RH. For comparison, in model media, viabilities varied by only an order of magnitude over the same RH range, which is consistent with past studies [8], [13]. Thus, RH might have a greater effect on IAV’s survival in mucus than has been demonstrated in past studies with synthetic media. These results underscore the potential impact of RH on the virus’ survival in its natural aerosol carrier during transmission.\n\nImplications for Influenza’s Transmission Patterns\nMany mechanisms have been proposed to explain influenza’s seasonality, including (1) environmental and climatic factors (e.g., temperature, relative or absolute humidity, and solar intensity), (2) host behavioral changes (e.g., school schedule and increased crowding during winter or rainy seasons), and (3) oscillations in host immunocompetence (e.g., vitamin D levels and melatonin levels) [7], [35]. A recent review by Tamerius et al. [7] assesses the feasibility of various mechanisms and concludes that the central questions in influenza seasonality remain unresolved. Our study was designed to focus on the effect of humidity, and we conducted all experiments at room temperature. In indoor environments, where infection is more likely to occur due to the much larger amount of time spent there and the greater spatial density of potential hosts, temperature tends to fall in a narrow range around 20°C. With this restriction, we were not able to distinguish between the effects of relative v. absolute humidity.\nOur findings in human mucus could help explain, at least in part, the transmission patterns of influenza. In temperate regions, wintertime heating reduces RH in the indoor environment to low levels, usually \u003c40% [36], [37]. Low RHs not only help preserve the viability of IAV but also enable IAV carrier aerosols to persist longer in air because of their smaller size and lower settling velocities that result from more vigorous evaporation [16]. Thus, transmission of influenza in temperate regions could be enhanced in winter primarily via the aerosol route. In tropical regions, high temperatures may suppress transmission, particularly through the aerosol route [38], [39]. However, lower temperatures and near-saturated RH during the rainy season create an opportunity for transmission via different mechanisms for large droplets v. very small aerosols. Large droplets would settle more quickly due to gravitation because they do not shrink as much at ∼100% RH (only to 93% of their original diameters at 99% RH, and 76% at 98% RH [16]). Once settled on a surface, they may serve as a reservoir for contract transmission [39], [40] since IAV is well preserved at ∼100% RH, as shown in this study. On the other hand, submicron aerosols such as those exhaled in human breath [41] would remain aloft, and thanks to the lower temperatures and suitable RHs for survival, transmission by these submicron droplets via the aerosol route might still be effective.\n\nLimitation and Directions for Future Study\nIn this study, droplets of 1 µL, rather than smaller aerosols [42], were used to simulate the interplay of humidity, droplet evaporation, solute concentrations in the droplet, and virus viability. It takes ∼10 min for such droplets to dry out completely at ∼50% RH, considerably longer than for much smaller droplets (e.g., \u003c1 s for a respiratory droplet 20 µm in diameter [17]). The legitimacy of extrapolating our results to aerosols expelled from human respiratory tract thus depends on whether the dynamics of evaporation are critical for IAV decay. Our results in model media are comparable to those conducted in aerosols [8], [13]. Harper [13] recovered 66–126% of IAV in aerosols 1 s after spraying, when evaporation was completed. The high recoveries indicate that the effect of the evaporation process itself on viral decay is negligible. Even if it were critical, such an effect was controlled for when comparing viral decay in the same type of medium versus RH, since all the droplets experienced a similar rate of evaporation. Nevertheless, verifying these results in aerosols is warranted.\nThis study proposes a new mechanistic basis for the effect of RH on IAV viability in droplets. However, it does not address the mechanism by which salts affect IAV viability at a molecular level, nor does it explain how the presence of proteins alters the relationship. In addition, the relationship in mucus differed slightly from that observed in model media. Given the complex composition and unique nature of mucus, mechanisms governing the relationship in mucus might differ from those in model media.\n\nConclusions\nThis study reports novel data on the response of IAV in droplets of model media to varying RH, including extreme conditions that have never been studied, and for the first time presents the relationship between IAV viability in human mucus and humidity over a large range of RH. Results suggest that there exist three regimes of IAV viability defined by RH. We provide a mechanistic explanation for these regimes, based on droplet evaporation, a subsequent increase in solute concentrations in the droplet, and their effect on the virus. Our theory also explains the conflicting findings in the literature about IAV viability in airborne droplets [8], [13]–[15]. We further outline a new perspective on the dependence of IAV’s transmission on humidity, which introduces a possible explanation for influenza’s seasonal patterns in different 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