Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice

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1. Version published to 10.1038/s41467-020-19891-7

   Nov 30, 2020
2. 

   ScreenIT

   Jul 20, 2020

   SciScore for 10.1101/2020.07.18.210179: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: All experimental procedures with animals were approved by the Texas Biomedical Research Institute (Texas Biomed) Institutional Biosafety Committee (IBC, #20-004 and #20-010) and Institutional Animal Care and Use Committee (IACUC, #1708 MU) and under Biosafety Level 3 (BSL3) and animal BSL3 (ABSL3) facilities at Texas Biomed.
   Randomization	not detected.
   Blinding	Sections were stained with Haemotoxylin and Eosin (H&E) and evaluated using light microscopy in a blinded manner by a board certified veterinary pathologist.
   Power Analysis	not detected.
   Sex as a biological variable	Mice: Specific-pathogen-free, 4-5-weeks-old, female and male …

   SciScore for 10.1101/2020.07.18.210179: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: All experimental procedures with animals were approved by the Texas Biomedical Research Institute (Texas Biomed) Institutional Biosafety Committee (IBC, #20-004 and #20-010) and Institutional Animal Care and Use Committee (IACUC, #1708 MU) and under Biosafety Level 3 (BSL3) and animal BSL3 (ABSL3) facilities at Texas Biomed.
   Randomization	not detected.
   Blinding	Sections were stained with Haemotoxylin and Eosin (H&E) and evaluated using light microscopy in a blinded manner by a board certified veterinary pathologist.
   Power Analysis	not detected.
   Sex as a biological variable	Mice: Specific-pathogen-free, 4-5-weeks-old, female and male B6.Cg-Tg(K18-ACE2)2Prlmn/J (Stock No: 034860, K18 hACE2) hemi-zygotes, or wild-type (WT) C57BL/6 control mice, were purchased from The Jackson Laboratory (Bar Harbor, ME).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The primary antibodies were detected with goat anti-rabbit-AP developed with Permanent Red warp (mach3 Biocare Medical).	anti-rabbit-AP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Measurement of viral loads: Confluent monolayers of Vero E6 cells (96-well plate format, 4 x 104 cells/well, duplicates) were infected with 10-fold serial dilutions of supernatants obtained from the organ homogenates.	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice: Specific-pathogen-free, 4-5-weeks-old, female and male B6.Cg-Tg(K18-ACE2)2Prlmn/J (Stock No: 034860, K18 hACE2) hemi-zygotes, or wild-type (WT) C57BL/6 control mice, were purchased from The Jackson Laboratory (Bar Harbor, ME).	B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860) C57BL/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   After incubation with the primary NP MAb, cells were washed three times with PBS, and developed with the Vectastain ABC kit and DAB Peroxidase Substrate kit (Vector Laboratory, Inc., CA, USA) according to the manufacturers’ instructions.	Vector Laboratory suggested: None
   Statistical analysis: Statistical significance was determined using Prism 8 software (GraphPad Software, San Diego, CA).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   While our findings clearly identify the K18 hACE2 transgenic mouse as a model for SARS-CoV-2 infection and COVID-19 disease, our studies have some limitations. The primary limitation was the number of mice that were available for study, due to limited supply from the vendor. We maximized our studies by performing two independent experiments that cross-validated each other in survival and pathogenesis results. Both studies showed considerable sensitivity of K18 hACE2 transgenic mice to SARS-CoV-2 infection. We chose a relatively high viral dose (MOI 1x105 PFU/mouse) that was capable of causing extensive disease, but we were unable to include additional time points to further understand the time course of immune responses (e.g. 1-DPI) or whether the chemokine and cytokine storm increased again at study endpoints (e.g. 5/6-DPI). Likewise, the limited number of K18 hACE2 transgenic mice did not allow us to use different viral doses to calculate the mouse lethal dose 50 (MLD50) of SARS-CoV-2. Inclusion of WT C57BL/6 control mice in our studies confirmed that mice are not naturally susceptible to infection with SARS-CoV-2 without experimental manipulation to express hACE2, further confirming that hACE2 is the receptor for SARS-CoV-2.56–59 Several groups are developing large animal models of SARS-CoV-2 infection and COVID-19, with a primary focus on nonhuman primates (NHP) models. Reports indicate that macaques develop self-limiting disease with a strong anamnestic responses (innate...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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   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.

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3. 

   Version published to 10.1101/2020.07.18.210179v1 on bioRxiv

   Jul 19, 2020