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.

An unusual outbreak of rotavirus G8P[8] gastroenteritis in adults in an urban community, Singapore, 2016.

BACKGROUND: An outbreak of gastroenteritis (GE) occurred in community-dwelling adults in the Central Region of urban Singapore, in May 2016. OBJECTIVES: To investigate the cause of the outbreak. STUDY DESIGN: We conducted a case-cohort study on GE patients linked to the outbreak who presented to the emergency department of a tertiary-care hospital near the outbreak area, from 18 May to 11 June 2016. Stools were tested for gastrointestinal pathogens including rotavirus antigen and positive rotavirus samples were subject to genotyping. RESULTS: A total of 57 adult GE patients, with a median age of 40 (range 18 to 84) years, were included. Predominant symptoms were diarrhoea (98.2%), vomiting (64.9%), and abdominal discomfort (38.6%). Age 65 years and above (Adjusted OR 21.78, 95% CI 1.49-318.84; P = 0.02) was the only predictor of admission, after adjusting for comorbidities and clinical severity. Molecular microbiological investigations confirmed that the outbreak was caused by a novel human-bovine reassortant strain of rotavirus G8P[8] with DS-1-like backbone. Exposure to the market in the outbreak area was strongly associated with rotavirus infection (OR 46.14; 95% CI 43.04-49.25, P < 0.01). No particular food item could be identified as the outbreak cause. CONCLUSIONS: This is the first report of an outbreak of rotavirus G8P[8] in adults in an urban community that is not waterborne. Transmission was likely through fomites in the market and its surrounding areas, via consumption or contact with contaminated food items purchased from the market, and from person-to-person. The potential for novel G8P[8] strains to cause outbreaks cannot be overemphasized.

Outbreak of caliciviruses in the Singapore military, 2015.

BACKGROUND: From 31 August to 9 September 2015, a total of 150 military personnel at a military institution in Singapore were infected with acute gastroenteritis (AGE) with an attack rate of approximately 3%. This study aimed to determine the epidemiology of the outbreak, investigate its origins, and discuss measures to prevent future occurrences. METHODS: After the AGE outbreak was declared on 31 August 2015, symptom surveys, hygiene inspections, and the testing of water, food, and stool samples were initiated. We collected 86 stool samples from AGE cases and 58 samples from food-handlers during the course of the outbreak and these stool samples were tested for 8 bacterial pathogens and 2 viral pathogens (i.e., norovirus and sapovirus). RESULTS: We detected Sapovirus (SaV), group I Norovirus (NoV GI) and group II Norovirus (NoV GII) from the stool samples of AGE cases. Further sequence analyses showed that the AGE outbreak in August was caused mainly by three rarely reported calicivirus novel genotypes: NoV GI.7, NoV GII.17 and SaV GII.3. Control measures implemented focused on the escalation of personal and environmental hygiene, which included the separation of affected and unaffected soldiers, enforcement of rigorous hand-washing and hygiene, raising awareness of food and water safety, and disinfection of communal areas with bleach. CONCLUSIONS: This study identified both NoV and SaV as the causative agents for an AGE outbreak at a Singapore military camp in August 2015. This study is also the first to report SaV as one of the main causative agents, highlighting the importance of caliciviruses as causative agents of AGE outbreaks in the Singapore military. As there are no commercially available vaccines against caliciviruses, strict personal hygiene and proper disinfection of environmental surfaces remain crucial to prevent calicivirus outbreak and transmission.

Emergence of norovirus GI.2 outbreaks in military camps in Singapore.

BACKGROUND: Simultaneous acute gastroenteritis (AGE) outbreaks occurred at two military camps. This study details the epidemiological findings, explores possible origins, and discusses preventive measures. METHODS: Investigations included attack rate surveys, symptom surveys, hygiene inspections, and the testing of water, food, and stool samples. DNA/RNA was extracted from stool samples and amplified via real-time reverse transcription PCR (RT-PCR). Partial and full-length capsid nucleotide sequences were obtained, phylogenetic relationships inferred, and homology modelling of antigenic sites performed. RESULTS: The military outbreaks involved 775 persons and were preceded by two AGE outbreaks at restaurants in the local community. The outbreak was longer and larger in the bigger camp (21 days, attack rate 15.0%) than the smaller camp (6 days, attack rate 8.3%). Of 198 stool samples, norovirus GI.2 was detected in 32.5% (larger camp) and 28.6% (smaller camp). These were essentially identical to preceding community outbreaks. Antigenic site homology modelling also showed differences between identified and more common AGE outbreak strains (norovirus GII.4). CONCLUSION: Differences observed highlight difficulties in controlling person-to-person outbreaks among large groups in close proximity (e.g., military trainees). Distinct differences in antigenic sites may have contributed to increased immunological susceptibility of the soldiers to infection.

Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.