Early detection of SARS-CoV-2 in circulating immune cells in a mouse model

Abstract

SARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope™ Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.

SciScore for 10.1101/2021.06.30.450531: (What is this?)

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

Table 1: Rigor

Ethics	IACUC: Mouse infection and sample collection: Mouse experiments were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee at Yale University.
Sex as a biological variable	8-10 weeks-old female C57BL/6J mice (JAX Stock #: 000664) were inoculated with 2×108 PFU of Ad5-hACE2 by intranasal instillation.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: These cell lines are not listed in the database of commonly misidentified cell lines maintained by ICLAC, and in our hands tested negative for mycoplasma contamination.

Table 2: Resources

Antibodies
Sentences	Resources
Signals for 5 fluorescent …

SciScore for 10.1101/2021.06.30.450531: (What is this?)

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

Table 1: Rigor

Ethics	IACUC: Mouse infection and sample collection: Mouse experiments were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee at Yale University.
Sex as a biological variable	8-10 weeks-old female C57BL/6J mice (JAX Stock #: 000664) were inoculated with 2×108 PFU of Ad5-hACE2 by intranasal instillation.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: These cell lines are not listed in the database of commonly misidentified cell lines maintained by ICLAC, and in our hands tested negative for mycoplasma contamination.

Table 2: Resources

Antibodies
Sentences	Resources
Signals for 5 fluorescent markers were created by staining with antibodies against the common leukocyte antigen (Rat-anti-mouse CD45, Cat# 550539, clone: 30-F11, BD Pharmingen, San Jose, CA 95131, USA) indirectly labeled with a goat-anti-rat secondary antibody labeled with Alexa Fluor 488, a dendritic cell anti-CD11c marker (Hamster-anti-Mouse CD11c, Cat# 553799, clone HL3, BD Pharmingen) indirectly labeled with goat-anti-Hamster Alexa Fluor 594, hACE2 (Mouse-anti-Human ACE-2, Cat# sc-390851, clone E-11, Santa Cruz Biotechnology) indirectly labeled with goat-anti-mouse Alexa Fluor 594, and the RNA-FISH Spike Probe fluorescently labeled with Quasar 670.	CD45 suggested: (BD Biosciences Cat# 550539, RRID:AB_2174426) secondary suggested: (RayBiotech Cat# DS-MB-00263, RRID:AB_852159) CD11c suggested: (BD Biosciences Cat# 553799, RRID:AB_395058) Alexa Fluor 594, hACE2 suggested: None ACE-2 suggested: None Alexa Fluor 594, suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell and virus culture: Vero cells (monkey kidney epithelial cells, Cat. # CCL-81) were purchased from ATCC (Manassas, VA, USA).	Vero suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
Experimental Models: Organisms/Strains
Sentences	Resources
8-10 weeks-old female C57BL/6J mice (JAX Stock #: 000664) were inoculated with 2×108 PFU of Ad5-hACE2 by intranasal instillation.	C57BL/6J suggested: RRID:IMSR_JAX:000664)
Software and Algorithms
Sentences	Resources
The 48 oligomers underwent basic local alignment search tool (BLAST) analysis to eliminate off-target hybridization.	BLAST suggested: (BLASTX, RRID:SCR_001653)
Utilizing proprietary script programs created on the Fiji/ImageJ software platform, 3-D image stacks of the immobilized cells are acquired, individually for each of the 5 fluorescent markers.	Fiji/ImageJ suggested: None

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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.
No funding statement was detected.
No protocol registration statement was detected.

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

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Early detection of SARS-CoV-2 in circulating immune cells in a mouse model

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