Intermediate Relative Humidity Preserves Respiratory Syncytial Virus via a Semi-Solid Bioaerosol State

Respiratory syncytial virus (RSV) transmission via the aerosol route remains poorly understood, particularly with respect to how evolving virus-laden particles (bioaerosols) microenvironments influence viral survival. Bioaerosol particles contain complex mixtures of organic and inorganic components, and their physicochemical properties change dynamically during evaporation as water is lost upon emission from respiratory activities. These changes directly affect the local environment surrounding embedded virus during both the evaporation stage and the subsequent equilibrium state. However, how these microenvironmental conditions under different relative humidity (RH) levels regulate RSV survival remains unclear. In this study, we quantified RSV survival during the evaporation and early equilibrium stages using a flow‑tube system with controlled residence times. Bioaerosols were generated from virus medium alone or supplemented with bovine serum albumin (BSA) or mucin and evaluated under low (35%) and intermediate (61%) RH conditions. Viral infectivity was normalized to RNA copy number to account for particle and sampling losses. At 35% RH, RSV infectivity decreased by one to three orders of magnitude, depending on the solution composition. In contrast, survival was significantly higher at intermediate RH, particularly for virus medium and BSA‑supplemented aerosols. Scanning electron microscopy revealed that low RH conditions promote efflorescence, whereas intermediate RH results in viscous or semi‑solid particles with higher water content. These observations suggest that efflorescence is associated with enhanced RSV inactivation, while viscous or semi‑solid phases tend to preserve RSV in the aerosol state for respirable particles. Overall, RSV infectivity depends strongly on particle chemical composition, phase state (effloresced versus semi‑solid), and relative humidity. These results highlight the importance of characterizing particle phase behavior and chemical composition during early aerosol processes to improve mechanistic understanding of viral survival relevant to short‑range transmission.