The size and culturability of patient-generated SARS-CoV-2 aerosol

Abstract

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.

Keywords: SARS-CoV-2; aerosol transmission; human-generated aerosol; viral aerosol.

Fig. 1. Measured Airborne RNA Concentrations and Associated Percent Change in Viral Copies in Cell Culture.

Measured rRT-PCR RNA copies/L of air (black) indicate in initial viral RNA concentrations while the change viral RNA (red) indicates viral replication in cell culture (or lack thereof). Error bars indicate the standard deviation of the RT-PCR-derived concentrations (for air concentration) and calculated measurement uncertainty (for percent change in cell culture). The P value comparing the RNA in cell culture supernatant on day 1 vs RNA on the final day is shown if the change is positive and the P value is <0.1. Three of the six submicron filter samples indicated an increase in cell culture that was significant (p < 0.05) based on the Students T Test (7B, 5A and 5C. Two of the 1–4 um samples had P values < 0.1, but not <0.05 (7A and 5C). None of >4.1 um samples demonstrated statistically significant replication. Samples with a ratio of <1 indicate a loss of RNA between the first and last day of culture. Cell culture controls (right hand side) indicate the results when known concentration of laboratory cultured virus are introduced, as well as extracted RNA.

Fig. 2. Protein expression of SARS-CoV-2.

Cell protein lysates were prepared from the indicated cultured samples and subsequently probed by western blot with a mouse monoclonal anti-SARS nucleocapsid protein (SARS-CoV N) antibody and an anti-GAPDH loading control antibody. The gel images between 40 and 55 kDa are shown. Images of control gels from infections initiated at titers from 10⁻² to 10² pfu/mL are shown in supplementary Fig. S1, for comparison.

Fig. 3. Electron micrographs of SARS-CoV-2 virions cultivated from the sub-micron filter from Room 5C.

The same image is shown at two magnifications: (A) ×30,000 and (B) ×110,000. Identifiable SARS-CoV-2 virions can be seen at both magnifications, and are indicated by red arrows in (B).