PMC:7723248 / 23130-40329 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":"Discussion\nOur results show that the chemistry of carrier droplets has significant impacts on the viability of both non-enveloped and enveloped viruses. The results suggest that the chemical composition of carrier droplets can influence the stability of viruses when they are released into the environment. While salt, pH, and surfactant reduced the viability of viruses at most RH conditions, protein provided some protection against virus decay in droplets. The effect of chemical composition was coupled with RH, which emphasizes the importance of exploring the effects of droplets’ chemical composition and environmental factors simultaneously in investigating the survival of viruses in the environment.\n\nSalt\nSodium chloride promoted the inactivation of viruses at low RH, but it did not affect, and sometimes even reduced, the decay of viruses at intermediate and high RHs. These seemingly conflicting results can be explained by two distinct mechanisms. Firstly, previous studies have reported that NaCl inactivates viruses, possibly by damaging viral RNA [48, 49], although the mechanism of inactivation has not been explicitly identified [48, 50]. Our results confirmed the effect, as decay of MS2 and Φ6 was enhanced in droplets containing NaCl at 20% RH. On the other hand, studies have suggested that viruses tend to aggregate in solutions with high salt concentration [51–54]. The formation of large virus aggregates increases virus stability in such environments [52]. Although other studies have demonstrated that low levels of salt (e.g., initial concentration of 1 g/L NaCl in our droplets) do not effectively facilitate the formation of virus aggregates [55, 56], we speculate that virus aggregation would be enhanced in evaporating droplets. This is because the salt concentration increases in evaporating droplets as they lose water, especially when they are close to desiccation, while the decrease in droplet volume brings viruses into contact with one another. We hypothesize that the increased relative viability of MS2 and Φ6 in droplets containing sodium chloride at 80% RH is due to the formation of virus aggregates. Further studies are needed to provide direct evidence supporting this hypothesis.\nThe observed RH-dependent effect of salt on the viability of viruses suggests that the relative contribution of the abovementioned mechanisms may vary at different RH conditions. The evaporation kinetics of droplets at various RHs seems to play an important role. Droplets with an initial volume of 1 μL evaporate rapidly at low RH (Fig 6A), desiccating within 15 minutes at 20% RH, whereas the evaporation process is much slower at high RH (Fig 6B and 6C). It is plausible that at low RH, droplets quickly desiccate before considerable amounts of virus aggregates are generated, in which case the inactivation effect of NaCl dominates and results in enhanced decay of viruses. Conversely, at high RH, droplet evaporation is much slower, allowing viruses to form aggregates and thus protecting viruses from inactivation. Again, additional investigation is needed to test this hypothesis.\nFig 6 Evaporation rate of droplets with different chemical compositions at (A) 20% RH, (B) 50% RH, and (C) 80% RH.\nThe lines represent the mean of duplicates. Error bars are not shown to facilitate visualization. The relative standard deviation averaged 7%, 18%, and 7% at 20%, 50%, and 80% RH, respectively.\n\npH\nOur results demonstrate that pH affects the stability of MS2 and Φ6 differently in droplets. MS2 survived equally well in acidic, pH-neutral, and basic droplets, whereas Φ6 survived best in pH-neutral droplets and decayed more in acidic or basic droplets. Previous studies have reported that viruses in bulk solutions are sensitive to pH [57, 58]. Both non-enveloped and enveloped viruses are generally more susceptible in acidic and basic solutions than in pH-neutral solutions [57]. At extreme pHs, viruses decay due to the denaturing of surface proteins and the hydrolysis of the viral genome [48, 59]. However, MS2 appears to be insensitive to pH. In a previous study, a moderate decay rate of ~0.5 log10 unit per day was observed for MS2 in bulk solutions at pH values of 4 and 10, and MS2 retained its viability when the solution was pH-neutral. The effect of pH on the viability of enveloped viruses is generally more noticeable than its effect on non-enveloped viruses [57], consistent with our observation of pronounced decay of Φ6 in acidic solutions across all RH levels. Besides the protein denaturing effect, the fusion of enveloped viruses’ membrane structure caused by extreme pH also leads to inactivation [48]. Low-pH treatment is widely used in monoclonal antibody purification processes to inactivate viruses because of its reliable performance (e.g., \u003e 4 log10 decay) on enveloped viruses [60].\nIn addition to the inactivation effect induced by pH, the dynamic change in pH of evaporating droplets can also affect virus survival. Although the pH of all virus suspensions was adjusted to the target pH at the beginning of experiments, the pH is likely to change as droplets evaporate. The loss of water will enrich ions, such as H3O+ and OH-, which may create pH-gradients inside droplets [46]. Additionally, since droplets were exposed to ambient air, the uptake of CO2 and formation of carbonic acid may lower the pH of droplets, but determining the extent of this process in evaporating droplets is challenging. Therefore, the pH of droplets is not expected to remain constant at its initial value throughout the experiment. The dynamic change in the pH of evaporating droplets introduces uncertainties in understanding its effect on the survival of viruses. Tools to monitor the real-time pH in evaporating droplets are necessary to fully explain the effect of pH on the viability of viruses in this complex system.\n\nProtein\nThe relative viability of MS2 and Φ6 was elevated in droplets containing BSA at RHs of 50% and 80%. Previous studies have found that the decay of viruses was greatly reduced in both aerosols and droplets supplemented with human respiratory fluid or fetal calf serum [17, 18]; protein may provide a protective effect. For example, influenza virus retained its viability in aerosols across a wide range of RHs after 1 h when the aerosolization media was supplemented with extracellular matrix from human bronchial epithelial cells. Here, we suspended viruses in media containing BSA and observed a similar protective effect. The detailed mechanism by which proteins protect viruses from decay remains unknown. Researchers have proposed that the inactivation of viruses in aerosols and droplets mainly happens at the air-water interface [61, 62]. The presence of proteins in droplets may reduce the solution surface tension, which inhibits viruses from reaching the air-water interface [63, 64]. Another possible mechanism is that potentially damaging compounds may first act on free proteins in droplets instead of those on the surfaces of viruses. Quantitative information on residual “free protein” in droplets over the course of exposure would be useful to test this hypothesis. It is also possible that proteins in solution may interact with those on the surface of viruses and help stabilize them.\n\nSurfactant\nSurfactants have been reported previously to enhance the inactivation of viruses [65, 66], which is in agreement with our results for Φ6. High concentrations of surfactant are very effective in inactivating enveloped viruses. For example, \u003e 4 log10 reduction has been reported after 1 h incubation in an 80 μM surfactin solution [67]. According to electron microscopy, the decay mechanism was concluded to be the disintegration of the lipid membrane and partial disintegration of the protein capsid on enveloped viruses. Since the initial concentrations of SDS in our droplets were much lower (3.4 and 34 μM), the magnitude of virus decay in our study was lower than previously reported.\nSince a lipid membrane is present only in enveloped viruses, the effect of surfactant on non-enveloped viruses is much weaker than on enveloped viruses [67]. Interestingly, we observed less decay of MS2 in droplets containing SDS compared to those without, suggesting a protective effect of SDS on the survival of non-enveloped viruses in droplets. Surfactants could protect viruses in a similar manner as proteins. Surfactants are known to strongly affect the surface tension of solutions, especially when the surfactant concentration is below the critical micelle concentration, beyond which micelles start to form and the surface tension of solutions remains relatively constant. Since the concentration of SDS examined in our study is much lower than its critical micelle concentration (8.2 mM), the presence of SDS in droplets could affect the surface tension and protect viruses from decay by hampering their ability to reach the air-water interface.\nTo our best knowledge, the effect of the chemical composition of droplets on the transmission of human or mammalian viruses has not been reported previously. Our results with model bacteriophages indicate that their survival in droplets is sensitive to the concentrations of different components in the droplets. This observation agrees with the findings from a previous study that investigated the effects of salt and protein on the survival of influenza virus in droplets; high salt concentrations were correlated with greater virus inactivation while protein protected the virus [17]. Since viruses must retain their infectivity to transmit successfully, our results imply that the chemical composition of droplets may influence virus transmission by modulating the survival of viruses.\n\nRelative humidity\nAn association between RH and virus transmission has been reported. For example, the incidence of influenza A in Hong Kong increased with higher RH, and the number of positive test results for influenza A was negatively correlated with RH in Singapore [68, 69]. Results from these epidemiological studies suggest that RH affects virus transmission. Furthermore, studies have demonstrated the effect of RH on the survival of human viruses (e.g., SARS-CoV-2 [70, 71]), underlining the importance of understanding the effect of RH on virus transmission.\nRH has large impacts on the viability of viruses in droplets, larger than the effect of chemical composition in some cases. We observed U-shaped patterns in the viability of MS2 against RH, and monotonically decreasing relationships between the viability of Φ6 and RH, respectively, in droplets of different compositions. We reported previously that the viability of MS2 and Φ6 in droplets composed of culture medium follows U-shaped patterns, in which the lowest viability occurs at 55% and 85% RH, respectively [13]. Many other studies have reported a similar pattern with greater decay at intermediate RHs than at lower or higher RHs [17, 18, 25, 27, 72]. The viability patterns observed in this study for Φ6, decreasing with RH rather than U-shaped, seem to conflict with results in the literature. However, we examined the viability of Φ6 between 20% and 80% RH in the current study. Over this range, the viability of viruses also decreased monotonically in previous studies; we have shown that the minimum viability of Φ6 in droplets occurs around 85% RH, beyond the range examined in the present study [13].\nAs we concluded in our prior study [13], RH affects the viruses’ viability mainly by controlling droplet evaporation kinetics, inducing changes in solute concentrations and the cumulative dose of harmful compounds to which viruses are exposed. At intermediate RH, the cumulative dose is higher because the solute concentrations increase relatively quickly and are then maintained at a high level throughout the experiment. While our previous work focused on viruses in their prescribed culture medium, results of the present study indicate that their viability follows the same pattern in droplets consisting of culture medium diluted 100x in ultrapure water and lacking salt, protein, and surfactant. Components in LB medium that are potentially harmful for viruses, though diluted, can accumulate as droplets evaporate and eventually cause virus inactivation over time.\nRH is the major factor that determines droplet evaporation kinetics, as shown in Fig 6. The initial evaporation rate was much higher at 20% RH than at 50% and 80%. At 20% and 50% RH, droplets fully desiccated in 1 h. At both conditions, the evaporation rates were relatively steady at the beginning (the first ~10 min and ~15 min for 20% and 50% RH, respectively), but later gradually decreased. However, at 80% RH the evaporation rate was more consistent throughout the experiment, and droplets did not fully evaporate within 1 h.\nBesides ambient RH, droplet composition can affect evaporation rates as well. At certain RH conditions, the evaporation kinetics varied with chemical composition and initial solute concentration. Droplets containing BSA generally evaporated faster than droplets containing other components at 20% and 50% RH. Droplets containing 1 g/L NaCl evaporated faster than those containing 35 g/L NaCl. A previous study demonstrated that the evaporation rates of droplets containing less than 5.8 g/L NaCl was almost two times higher than for droplets containing 58 g/L NaCl at RH \u003c 60% [73]. The authors concluded that Marangoni flows induced by surface tension gradients, which originated from local peripheral salt enrichment, caused the difference in evaporation rate. We observed that droplets with higher initial SDS concentration evaporated slower than those with lower initial SDS concentration at RHs of 20% and 50%. However, the result was completely the opposite at 80% RH, at which droplets containing 10 μg/mL SDS evaporated significantly faster than those containing 1 μg/mL SDS. Droplets containing different initial BSA concentrations and at different initial pH values had similar evaporation rates across all RH levels. Since the evaporation kinetics determine the change in solute concentrations and cumulative dose, it is necessary to understand the influence of droplet chemical composition and concentrations on the evaporation rates of virus-containing droplets.\n\nLimitations\nWhile this study provides novel results on the viability of viruses in evaporating droplets of different compositions over a range of RHs, it does not examine how the surface material upon which viruses are deposited might affect viability. Previous studies have reported that the persistence of viruses in droplets depends on the type of material (e.g., plastic vs. steel) [74]. It is possible that material exchange between surfaces and droplets (e.g., dissolution of metal ions into droplets) leads to accumulation of surface materials in droplets and inactivates viruses. It will be interesting to investigate the effects of the interplay among surface materials, droplet composition, and environment on the survival of viruses in droplets. Additionally, we have focused on the biological inactivation of viruses in droplets but not on their physical behavior, which likely depends on physicochemical characteristics of the droplets. Future studies should be conducted in this area, although pinpointing viruses within droplets is challenging. Results might help explain the protective or inactivating effect of certain media components we observed in this study. Previous studies have demonstrated differences in the persistence of viruses on surfaces as a function of initial viral titer in the inoculum [75, 76]. Investigating the role of viral titer, which might affect aggregation and other characteristics, on virus survival is another interesting research question. Lastly, this study reported findings from two bacteriophage models, which may not fully represent human viruses. Human viruses, such as influenza virus and coronavirus, should be used in future studies to elucidate the effects of droplets’ chemical composition and RH on virus survival and transmission of viruses.\nTo conclude, we demonstrated that both the chemical composition of droplets and RH strongly affect the viability of non-enveloped and enveloped viruses. The effects of sodium chloride and SDS varied by RH level and virus type. pH did not affect the viability of MS2 but effectively inactivated Φ6 in solutions at pHs of 4 and 10. BSA generally preserved the viability of MS2 and Φ6 in droplets. We also found that the viability of viruses in droplets of certain compositions was RH-dependent at most conditions. Our results reveal that two factors contribute to the inactivation of viruses in droplets: (1) droplet evaporation kinetics, which are controlled by RH; and (2) inactivation or protective effects induced by chemicals. Additionally, the physical behavior of viruses, such as forming aggregates and partitioning to the air-liquid interface, resulting from changes in droplets’ characteristics may also affect inactivation. Results from our study are meaningful in predicting the persistence of viruses in droplets of various compositions in the environment and infectious disease transmission.","divisions":[{"label":"title","span":{"begin":0,"end":10}},{"label":"p","span":{"begin":11,"end":708}},{"label":"sec","span":{"begin":710,"end":3425}},{"label":"title","span":{"begin":710,"end":714}},{"label":"p","span":{"begin":715,"end":2227}},{"label":"p","span":{"begin":2228,"end":3115}},{"label":"figure","span":{"begin":3116,"end":3425}},{"label":"label","span":{"begin":3116,"end":3121}},{"label":"caption","span":{"begin":3123,"end":3425}},{"label":"title","span":{"begin":3123,"end":3231}},{"label":"p","span":{"begin":3232,"end":3425}},{"label":"sec","span":{"begin":3427,"end":5868}},{"label":"title","span":{"begin":3427,"end":3429}},{"label":"p","span":{"begin":3430,"end":4844}},{"label":"p","span":{"begin":4845,"end":5868}},{"label":"sec","span":{"begin":5870,"end":7278}},{"label":"title","span":{"begin":5870,"end":5877}},{"label":"p","span":{"begin":5878,"end":7278}},{"label":"sec","span":{"begin":7280,"end":9725}},{"label":"title","span":{"begin":7280,"end":7290}},{"label":"p","span":{"begin":7291,"end":7978}},{"label":"p","span":{"begin":7979,"end":8935}},{"label":"p","span":{"begin":8936,"end":9725}},{"label":"sec","span":{"begin":9727,"end":14290}},{"label":"title","span":{"begin":9727,"end":9744}},{"label":"p","span":{"begin":9745,"end":10295}},{"label":"p","span":{"begin":10296,"end":11410}},{"label":"p","span":{"begin":11411,"end":12282}},{"label":"p","span":{"begin":12283,"end":12814}},{"label":"p","span":{"begin":12815,"end":14290}},{"label":"title","span":{"begin":14292,"end":14303}},{"label":"p","span":{"begin":14304,"end":16095}}],"tracks":[]}