ABSTRACT The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene ( SFTPC ) and the alveolar macrophage marker gene ( MARCO ) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene ( AGER ). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening.
【저자키워드】 Single Cell, Cellular imaging, RNA FISH, fluorescent image analysis, 【초록키워드】 COVID-19, coronavirus disease, SARS-CoV-2, pathology, Placenta, Infection, lung, Drug screening, Autopsy, diagnostics, RNA, amplification, Region, FFPE, Cell culture, SARS-CoV-2 RNA, Viral RNA, specimens, platform, AT1, AT2, Lymph node, fixation, Alveolar macrophages, formalin, tissue, infected patients, tissues, subgenomic mRNAs, novel coronavirus SARS-CoV-2, ORF1a, viral life cycle, cell line, Marco, subcellular localization, human lung tissue, SFTPC, alveolar, probe, regions, widespread, Cell, marker genes, marker gene, observé, identify, performed, caused, human tissue, subcellular, AGER, infected with SARS-CoV-2, macrophage marker, paraffin embedding, 【제목키워드】 Human, detection, SARS-CoV-2 RNA, Localization, pneumocyte, Type,