The Cellular Characterization of SARS-CoV-2 Spike Protein in Virus-Infected Cells Using the Receptor Binding Domain Binding Specific Human Monoclonal Antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23, and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 spike (S) protein, only PD5, PD7, and SC23 were able to bind to the receptor binding domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localized within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD-binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognized the uncleaved S protein, indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining were demonstrated for each isolate, SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. IMPORTANCE The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore, an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS- CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID-19 patients to monitor the RBD in cells infected with SARS-CoV-2 clinical isolates. These immunological reagents specifically recognize the correctly folded RBD and were used to monitor the appearance of the RBD in SARS-CoV-2-infected cells and identified the site where the RBD first appears.

Lack of viable severe acute respiratory coronavirus virus 2 (SARS-CoV-2) among PCR-positive air samples from hospital rooms and community isolation facilities.

BACKGROUND: Understanding the extent of aerosol-based transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for tailoring interventions for control of the coronavirus disease 2019 (COVID-19) pandemic. Multiple studies have reported the detection of SARS-CoV-2 nucleic acid in air samples, but only one study has successfully recovered viable virus, although it is limited by its small sample size. OBJECTIVE: We aimed to determine the extent of shedding of viable SARS-CoV-2 in respiratory aerosols from COVID-19 patients. METHODS: In this observational air sampling study, air samples from airborne-infection isolation rooms (AIIRs) and a community isolation facility (CIF) housing COVID-19 patients were collected using a water vapor condensation method into liquid collection media. Samples were tested for presence of SARS-CoV-2 nucleic acid using quantitative real-time polymerase chain reaction (qRT-PCR), and qRT-PCR-positive samples were tested for viability using viral culture. RESULTS: Samples from 6 (50%) of the 12 sampling cycles in hospital rooms were positive for SARS-CoV-2 RNA, including aerosols ranging from <1 µm to >4 µm in diameter. Of 9 samples from the CIF, 1 was positive via qRT-PCR. Viral RNA concentrations ranged from 179 to 2,738 ORF1ab gene copies per cubic meter of air. Virus cultures were negative after 4 blind passages. CONCLUSION: Although SARS-CoV-2 is readily captured in aerosols, virus culture remains challenging despite optimized sampling methodologies to preserve virus viability. Further studies on aerosol-based transmission and control of SARS-CoV-2 are needed.

Environmental contamination in a coronavirus disease 2019 (COVID-19) intensive care unit-What is the risk?

BACKGROUND: The risk of environmental contamination by severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in the intensive care unit (ICU) is unclear. We evaluated the extent of environmental contamination in the ICU and correlated this with patient and disease factors, including the impact of different ventilatory modalities. METHODS: In this observational study, surface environmental samples collected from ICU patient rooms and common areas were tested for SARS-CoV-2 by polymerase chain reaction (PCR). Select samples from the common area were tested by cell culture. Clinical data were collected and correlated to the presence of environmental contamination. Results were compared to historical data from a previous study in general wards. RESULTS: In total, 200 samples from 20 patient rooms and 75 samples from common areas and the staff pantry were tested. The results showed that 14 rooms had at least 1 site contaminated, with an overall contamination rate of 14% (28 of 200 samples). Environmental contamination was not associated with day of illness, ventilatory mode, aerosol-generating procedures, or viral load. The frequency of environmental contamination was lower in the ICU than in general ward rooms. Eight samples from the common area were positive, though all were negative on cell culture. CONCLUSION: Environmental contamination in the ICU was lower than in the general wards. The use of mechanical ventilation or high-flow nasal oxygen was not associated with greater surface contamination, supporting their use and safety from an infection control perspective. Transmission risk via environmental surfaces in the ICUs is likely to be low. Nonetheless, infection control practices should be strictly reinforced, and transmission risk via droplet or airborne spread remains.