PMID- 35130722 OWN - NLM STAT- MEDLINE DCOM- 20230228 LR - 20230307 IS - 2150-7511 (Electronic) VI - 13 IP - 1 DP - 2021 Feb 22 TI - Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages. PG - e0375121 LID - 10.1128/mbio.03751-21 [doi] LID - e03751-21 AB - 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. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells. FAU - Acheampong, Kofi K AU - Acheampong KK AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Schaff, Dylan L AU - Schaff DL AD - Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Emert, Benjamin L AU - Emert BL AD - Genomics and Computational Biology Graduate Group, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Lake, Jonathan AU - Lake J AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Reffsin, Sam AU - Reffsin S AD - Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Shea, Emily K AU - Shea EK AD - Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Comar, Courtney E AU - Comar CE AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Department of Microbiology, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Philadelphia, Pennsylvania, USA. AD - Clinical Microbiology Laboratory, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA. AD - Infectious Disease Diagnostics Laboratory, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. FAU - Litzky, Leslie A AU - Litzky LA AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Khurram, Nigar A AU - Khurram NA AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Department of Pathology, Northwestern Universitygrid.16753.36, Feinberg School of Medicine, Chicago, Illinois, USA. FAU - Linn, Rebecca L AU - Linn RL AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Division of Anatomic Pathology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. FAU - Feldman, Michael AU - Feldman M AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Weiss, Susan R AU - Weiss SR AUID- ORCID: 0000-0002-8155-4528 AD - Department of Microbiology, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Philadelphia, Pennsylvania, USA. FAU - Montone, Kathleen T AU - Montone KT AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Cherry, Sara AU - Cherry S AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Philadelphia, Pennsylvania, USA. AD - Department of Biochemistry and Biophysics, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. FAU - Shaffer, Sydney M AU - Shaffer SM AUID- ORCID: 0000-0003-3539-9886 AD - Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. AD - Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA. LA - eng GR - R01 AI152362/AI/NIAID NIH HHS/United States GR - DP5 OD028144/OD/NIH HHS/United States GR - R01 AI140539/AI/NIAID NIH HHS/United States GR - F30 CA236129/CA/NCI NIH HHS/United States GR - R01 AI150246/AI/NIAID NIH HHS/United States GR - R01 AI140442/AI/NIAID NIH HHS/United States GR - T32 HG000046/HG/NHGRI NIH HHS/United States GR - F30 AI114475/AI/NIAID NIH HHS/United States GR - T32 GM007170/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20220208 PL - United States TA - mBio JT - mBio JID - 101519231 RN - 0 (RNA, Viral) SB - IM MH - Humans MH - *Alveolar Epithelial Cells MH - Macrophages, Alveolar MH - SARS-CoV-2 MH - *COVID-19 MH - RNA, Viral MH - In Situ Hybridization, Fluorescence MH - Lung/pathology PMC - PMC8822351 OTO - NOTNLM OT - RNA FISH OT - cellular imaging OT - fluorescent image analysis OT - single cell COIS- The authors declare a conflict of interest. S.M.S. receives royalties related to Stellaris RNA FISH probes. All other authors declare no competing interests. S.M.S. receives royalties related to Stellaris RNA FISH probes. All other authors declare no competing interests. EDAT- 2022/02/09 06:00 MHDA- 2023/03/03 06:00 PMCR- 2022/02/08 CRDT- 2022/02/08 05:37 PHST- 2022/02/08 05:37 [entrez] PHST- 2022/02/09 06:00 [pubmed] PHST- 2023/03/03 06:00 [medline] PHST- 2022/02/08 00:00 [pmc-release] AID - 03751-21 [pii] AID - mbio.03751-21 [pii] AID - 10.1128/mbio.03751-21 [doi] PST - ppublish SO - mBio. 2021 Feb 22;13(1):e0375121. doi: 10.1128/mbio.03751-21. Epub 2022 Feb 8.