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Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques.

ABSTRACT: Low and persistent levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus can be detected in clinical samples months after infection, possibly related to the emergence of SARS-CoV-2 variants or development of long coronavirus disease. Here, we present a protocol to detect low levels of viral RNA together with protein using flow cytometry and microscopy. We describe steps for cell infection with SARS-CoV-2 and quantification by fluorescence in situ hybridization-flow cytometry. We then detail procedures for visualization using immunolabeling and RNAscope. This approach is directly applicable to clinical samples. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).¹.

SUBMITTER: Cottignies-Calamarte A

PROVIDER: S-EPMC10520661 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques.

Cottignies-Calamarte Andréa A He Feifan F Zhu Aiwei A Real Fernando F Bomsel Morgane M

STAR protocols 20230921 4

Low and persistent levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus can be detected in clinical samples months after infection, possibly related to the emergence of SARS-CoV-2 variants or development of long coronavirus disease. Here, we present a protocol to detect low levels of viral RNA together with protein using flow cytometry and microscopy. We describe steps for cell infection with SARS-CoV-2 and quantification by fluorescence in situ hybridization- ...[more]

PMID: 37738115

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Project description:The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). 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, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. 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, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. 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.

Dataset Information

Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques.

Publications

Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques.

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