Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization

Abstract

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.

SciScore for 10.1101/2021.08.11.455959: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: All work with SARS-CoV-2 was performed in a biosafety level 3 laboratory using appropriate personal protective equipment and protocols approved by the Institutional Biosafety Committee and Environmental Health and Safety at the University of Pennsylvania.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Cell lines and infection: We cultured A549ACE2 cells at 37°C and 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.	A549ACE2 suggested: None
We cultured Huh7.5 cells at 37°C …

SciScore for 10.1101/2021.08.11.455959: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: All work with SARS-CoV-2 was performed in a biosafety level 3 laboratory using appropriate personal protective equipment and protocols approved by the Institutional Biosafety Committee and Environmental Health and Safety at the University of Pennsylvania.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Cell lines and infection: We cultured A549ACE2 cells at 37°C and 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.	A549ACE2 suggested: None
We cultured Huh7.5 cells at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-Glutamax.	Huh7.5 suggested: RRID:CVCL_7927)
Software and Algorithms
Sentences	Resources
We divided each 52-mer oligo into two non-overlapping 25-mer sequences (removing the middle two nucleotides) and appended split-initiator HCR sequences using a custom matlab script (see Supp.	matlab suggested: (MATLAB, RRID:SCR_001622)
To target SARS-CoV-2 subgenomic RNAs, we referenced the UCSC Genome Browser for SARS-CoV-2 genome datasets (https://genome.ucsc.edu/covid19.html) and RNA-sequencing datasets44 to identify the most frequent junction locations and peri-junction sequences based on the most abundant sgRNA junction spanning reads.	UCSC Genome Browser suggested: (UCSC Genome Browser, RRID:SCR_005780)
We then used CellProfiler to segment cells using 4’,6-diamidino-2-phenylindole (DAPI) to identify nuclei.	CellProfiler suggested: None

Results from OddPub: Thank you for sharing your code.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization

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