Survival of MS2 and Φ6 viruses in droplets as a function of relative humidity, pH, and salt, protein, and surfactant concentrations

Abstract

The survival of viruses in droplets is known to depend on droplets' chemical composition, which may vary in respiratory fluid between individuals and over the course of disease. This relationship is also important for understanding the persistence of viruses in droplets generated from wastewater, freshwater, and seawater. We investigated the effects of salt (0, 1, and 35 g/L), protein (0, 100, and 1000 μg/mL), surfactant (0, 1, and 10 μg/mL), and droplet pH (4.0, 7.0, and 10.0) on the viability of viruses in 1-μL droplets pipetted onto polystyrene surfaces and exposed to 20%, 50%, and 80% relative humidity (RH) using a culture-based approach. Results showed that viability of MS2, a non-enveloped virus, was generally higher than that of Φ6, an enveloped virus, in droplets after 1 hour. The chemical composition of droplets greatly influenced virus viability. Specifically, the survival of MS2 was similar in droplets at different pH values, but the viability of Φ6 was significantly reduced in acidic and basic droplets compared to neutral ones. The presence of bovine serum albumin protected both MS2 and Φ6 from inactivation in droplets. The effects of sodium chloride and the surfactant sodium dodecyl sulfate varied by virus type and RH. Meanwhile, RH affected the viability of viruses as shown previously: viability was lowest at intermediate to high RH. The results demonstrate that the viability of viruses is determined by the chemical composition of carrier droplets, especially pH and protein content, and environmental factors. These findings emphasize the importance of understanding the chemical composition of carrier droplets in order to predict the persistence of viruses contained in them.

Fig 1. Factors that affect the survival of viruses in stationary droplets on surfaces.

Fig 2

Concentration of bacteriophages (A) MS2 and (B) Φ6 in droplets with different initial sodium chloride concentration before (dark bars) and after (light bars) 1 h exposure to low, intermediate, and high RH (mean ± s.d. of triplicates). Relative viability of (C) MS2 and (D) Φ6 after 1 h exposure (lines shown the mean of triplicates). The number of virions in droplets at the start of the exposure experiments was 10⁵−10⁶ PFU.

Fig 3

Concentration of bacteriophages (A) MS2 and (B) Φ6 in droplets with different initial pH values before (dark bars) and after (light bars) 1 h exposure to low, intermediate, and high RH (mean ± s.d. of triplicates). Relative viability of (C) MS2 and (D) Φ6 after 1 h exposure (lines show the mean of triplicates). The number of virions in droplets at the start of the exposure experiments was 10⁵−10⁶ PFU. The dark gray dashed line indicates the detection limit (10⁴ PFU/mL) of plaque assay. ND indicates no viable virus was detected.

Fig 4

Concentration of bacteriophages (A) MS2 and (B) Φ6 in droplets with different initial protein concentration before (dark bars) and after (light bars) 1 h exposure to low, intermediate, and high RH (mean ± s.d. of triplicates). Relative viability of (C) MS2 and (D) Φ6 after 1 h exposure (lines show the mean of triplicates). The number of virions in droplets at the start of the exposure experiments was 10⁵−10⁶ PFU.

Fig 5

Concentration of bacteriophages (A) MS2 and (B) Φ6 in droplets with different initial surfactant concentration before (dark bars) and after (light bars) 1 h exposure to low, intermediate, and high RH (mean ± s.d. of triplicates). Relative viability of (C) MS2 and (D) Φ6 after 1 h exposure (lines show the mean of triplicates). The number of virions in droplets at the start of the exposure experiments was 10⁵−10⁶ PFU. The dark gray dashed line indicates the detection limit (10⁴ PFU/mL) of plaque assay. ND indicates no viable virus was detected.

Fig 6. Evaporation rate of droplets with different chemical compositions at (A) 20% RH, (B) 50% RH, and (C) 80% RH.

The 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.