mRNA-FISH for the detection of viral RNA in SARS-CoV-2 infected cells and the determination of the kinetics of early RNA replication biorxivpreprint EinsteinMed IcahnMountSinai RNA SARSCoV2 infection covid coronavirus COVID19
By Tarun Sai LomteDec 15 2022Reviewed by Danielle Ellis, B.Sc. In a recent study posted to bioRxiv*, researchers evaluated the early replication kinetics of severe acute respiratory syndrome coronavirus 2 .
The study and findings The present study performed a time course analysis to evaluate SARS-CoV-2 replication kinetics. First, the researchers employed a single-molecule RNA fluorescence in situ hybridization technique to detect viral gRNA and sgRNA at a single-cell and -molecule level. As such, 40 fluorescent-labeled anti-sense oligonucleotides were developed based on spike gene sequence.
The SARS-CoV-2-infected cells were subjected to smRNA-FISH and visualized at 20x magnification using high-speed high-resolution scanning fluorescence microscopy . The authors observed small plaques characterized by probe-positive fluorescent clusters of cells around dead cells, indicating that the probe was selective for viral RNA in live infected cells.
At 6 hpi, spots representing single molecules and larger patches of multiple RNAs were evident. By 12 hpi, SARS-CoV-2 RNA filled the entire cytoplasm. Next, the authors evaluated the ability of another probe derived from non-structural protein 12 gene sequences to detect gRNA alone since P1 could detect both gRNA and sgRNA.
Further microscopic examinations suggested that individual positive spots at < 2 hpi were likely single gRNAs in each cell. At later time points, the larger spots represented ROs with gRNA and sgRNA in the center and the periphery, respectively. This was followed by the formation of multiple daughter ROs at later time points.
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