Replication of SARS-CoV-2 in human respiratory epithelium
cell biology imaging molecular biology
Authors: Milewska A. et al.
Link to paper: https://doi.org/10.1101/2020.03.20.999029.
Journal/ Pre-Print: bioRxiv
Key Words: COVID-19; SARS-CoV2; human airway epithelium; RNA-FISH;
Tag · Cell Biology · Molecular Biology · Imaging
1. SARS-CoV-2 actively replicates in human airway epithelial (HAE) cultures obtained from conductive airways, resected from transplant patients.
2. Development of RNA Fluorescent in situ Hybridisation (RNA-FISH) assay to visualise the infection in the tissue by confocal microscopy.
3. SARS-CoV-2 infects ciliated cells of the HAE.
Studies of SARS-CoV-2 are mostly carried out on the Vero E6 cell line that is a convenient model for testing anti-viral drugs, but limits the understanding of viral biology in human cells. Milewska et al. demonstrate that HAE cultures can provide a valuable tool for studying the virus in its natural microenviroment. HAE cultures inoculated with SARS-CoV-2 release virus particles from the apical side and virus titre reaches a plateau 48 hours post inoculation. Sub-genomic (sg) viral mRNA is abundant in the infected HAE cultures. The authors show that SARS-CoV-2 infects ciliated cells of the HAE employing a newly-developed RNA-FISH assay, which visualises viral RNA by confocal microscopy.
Impact for SARS-CoV2/COVID19 research efforts
Understand the virology and/or cell biology of SARS-CoV-2/COVID19: SARS-CoV-2 infects ciliate cells of the human airway epithelium.
Develop diagnostic tools for SARS-CoV-2/COVID19: RNA-FISH assay could be used to visualise infected cells in cultures reconstituted from patient samples.
· In vitro study
Strengths and limitations of the paper
Novelty: RNA-FISH assay to visualise infected cells.
Standing in the field: The use of HAE cultures to study coronaviruses is not novel. It has been reported that HAE provides a useful model to study SARS-CoV that infects ciliated cells and is shed towards the luminal surface (Sims A. et al, 2005). More recently, HAE were used to test the antiviral activity of a ribonucleoside analog against SARS-CoV-2 (Sheahan et al. 2020)
Appropriate statistics: Not applicable.
Viral model used: SARS-CoV-2 and Human Airway Epithelial (HAE) cultures.
Translatability: RNA-FISH could be developed as a rapid diagnostic tool to identify viral RNA in a tissue sample.
Main limitations: It is not clear how many times the authors repeated quantification of the sg mRNA of SARS-CoV-2-infected HAE cultures. It would have been nice to see a quantification of sg mRNA and whether its amount would fluctuate a lot within different replicates. It is not specified how many times the authors carried out the RNA-FISH assay. They have also mislabelled figures (Fig3A should be Fig 3B and vice versa). It is unclear what a mock control is for viral infection in Fig 3. It would have been more suitable to compare areas of the HAE culture with similar number of ciliated cells. Antibody used for cilia staining is not widely used and not typical for the field.