The Size and Culturability of Patient-Generated SARS-CoV-2 Aerosol

BACKGROUND: Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. OBJECTIVE: We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. METHODS: Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. SIGNIFICANCE: Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission

Fluorescence spectra and biological activity of aerosolized bacillus spores and MS2 bacteriophage exposed to ozone at different relative humidities in a rotating drum

AbstractBiological aerosols (bioaerosols) released into the environment may undergo physical and chemical transformations when exposed to atmospheric constituents such as solar irradiation, reactive oxygenated species, ozone, free radicals, water vapor and pollutants. Aging experiments were performed in a rotating drum chamber subjecting bioaerosols, Bacillus thuringiensis Al Hakam (BtAH) spores and MS2 bacteriophages to ozone at 0 and 150ppb, and relative humidities (RH) at 10%, 50%, and 80+%. Fluorescence spectra and intensities of the aerosols as a function of time in the reaction chamber were measured with a single particle fluorescence spectrometer (SPFS) and an Ultra-Violet Aerodynamic Particle Sizer® Spectrometer (UV-APS). Losses in biological activity were measured by culture and quantitative polymerase chain reaction (q-PCR) assay. For both types of aerosols the largest change in fluorescence emission was between 280 and 400nm when excited at 263nm followed by fluorescence emission between 380 and 700nm when excited at 351nm. The fluorescence for both BtAH and MS2 were observed to decrease significantly at high ozone concentration and high RH when excited at 263nm excitation. The decreases in 263nm excited fluorescence are indicative of hydrolysis and oxidation of tryptophan in the aerosols. Fluorescence measured with the UV-APS (355-nm excitation) increased with time for both BtAH and MS2 aerosols. A two log loss of MS2 bacteriophage infectivity was observed in the presence of ozone at ~50% and 80% RH when measured by culture and normalized for physical losses by q-PCR. Viability of BtAH spores after exposure could not be measured due to the loss of genomic material during experiments, suggesting degradation of extracelluar DNA attributable to oxidation. The results of these studies indicate that the physical and biological properties of bioaerosols change significantly after exposure to ozone and water vapor

Effects of ozone and relative humidity on fluorescence spectra of octapeptide bioaerosol particles

AbstractThe effects of ozone and relative humidity (RH) at common atmospheric levels on the properties of single octapeptide bioaerosol particles were studied using an improved rotating reaction chamber, an aerosol generator, an ultraviolet aerodynamic particle sizer (UVAPS), an improved single particle fluorescence spectrometer (SPFS), and equipments to generate, monitor and control the ozone and RH. Aerosol particles (mean diameter ~2μm) were generated from a slurry of octapeptide in phosphate buffered saline, injected into the rotating chamber, and kept airborne for hours. Bioaerosols were sampled from the chamber hourly for the measurements of particle-size distribution, concentration, total fluorescence excited at 355-nm, and single particle fluorescence spectra excited at 266-nm and 351-nm under different controlled RH (20%, 50%, or 80%) and ozone concentration (0 or 150ppb). The results show that: (1) Particle size, concentration, and the 263-nm-excited fluorescence intensity decrease at different rates under different combinations of the RH and ozone concentrations used. (2) The 263-nm-excited UV fluorescence (280–400nm) decreased more rapidly than the 263-nm-excited visible fluorescence (400–560nm), and decreased most rapidly when ozone is present and RH is high. (3) The UV fluorescence peak near 340nm slightly shifts to the shorter wavelength (blue-shift), consistent with a more rapid oxidation of tryptophan than tyrosine. (4) The 351/355-nm-excited fluorescence (430–580nm/380–700nm) increases when ozone is present, especially when the RH is high. (5) The 351/355-nm-excited fluorescence increase that occurs as the tryptophan emission in the UV decreases, and the observation that these changes occur more rapidly at higher RH with the present of ozone, are consistent with the oxidation of tryptophan by ozone and the conversion of the resulting ozonides to N-formyl kynurenine and kynurenine