A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories

Article activity feed

1. 

   ScreenIT

   Jun 3, 2021

   SciScore for 10.1101/2021.05.28.446200: (What is this?)

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

   Table 1: Rigor

   Ethics

   IACUC: Mouse strains: The animal’s experimental procedures were carried out with mixed groups (males and females) of mice aged 6 to 7 weeks and received the approval of the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (UFMG) (process No. 190/2020). Euthanasia Agents: MHV-3 infection: Mice were lightly anesthetized with an intraperitoneal injection of ketamine (50 mg/kg): xylazine (5 mg/kg) and received an intranasal inoculation of 30 μl sterile saline solution loaded or not (Mock controls) with MHV-3 at different concentrations (3×101 to 3×104 PFU).

   Sex as a biological variable

   Mouse strains: The animal’s experimental procedures were carried out with …

   SciScore for 10.1101/2021.05.28.446200: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mouse strains: The animal’s experimental procedures were carried out with mixed groups (males and females) of mice aged 6 to 7 weeks and received the approval of the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (UFMG) (process No. 190/2020). Euthanasia Agents: MHV-3 infection: Mice were lightly anesthetized with an intraperitoneal injection of ketamine (50 mg/kg): xylazine (5 mg/kg) and received an intranasal inoculation of 30 μl sterile saline solution loaded or not (Mock controls) with MHV-3 at different concentrations (3×101 to 3×104 PFU).
   Sex as a biological variable	Mouse strains: The animal’s experimental procedures were carried out with mixed groups (males and females) of mice aged 6 to 7 weeks and received the approval of the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (UFMG) (process No. 190/2020).
   Randomization	not detected.
   Blinding	The inflammation-mediated injury in mouse lungs was determined by a pathologist (C.M.Q.J) blinded to the experiment, by employing a scoring system encompassing: (i) airway inflammation (up to 4 points); (ii) vascular inflammation (up to 4 points); (iii) parenchyma inflammation (up to 5 points); and general neutrophil infiltration (up to 5 points) (40).
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Following permeabilization in PBS / 0,5% Triton X-100, the cryosections were incubated for 1 h in the blocking solution (PBS containing 5% goat serum and 5 μg/mL mouse BD Fc Block™) and then labeled overnight at 4°C with the APC-conjugated rat anti-mouse CD45 antibody (1:100 dilution, BD Pharmingen, cat No. 559864).	anti-mouse CD45 suggested: None
   1×106 cells were blocked with mouse BD FC block™ (5 μg/mL, BD Pharmingen, Cat No. 553141) and then stained using fluorescent-labeled monoclonal antibodies as follows: Ly6G-BV421 (1:200, BioLegend, Cat No. 127627); CD45-FITC (1:200	Ly6G-BV421 suggested: None
   The pre-synaptic and post-synaptic terminals were stained using the monoclonal anti-synaptotagmin antibody (1:250 dilution, Developmental Studies Hybridoma Bank; cat No. 3H2 2D7) and the tetramethylrhodamine-conjugated α-bungarotoxin (1:1000 dilution; Invitrogen, cat No. T1175), respectively.	anti-synaptotagmin suggested: (Thermo Fisher Scientific Cat# PA1-46358, RRID:AB_1090909) α-bungarotoxin suggested: (Thermo Fisher Scientific Cat# B35451, RRID:AB_2617152)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells, viruses and plaque assay: Vero E6 (ATCC® CRL-1586), L929 (ATCC® CCL-1), and Calu-3 (ATCC® HTB-55) cells were cultured under a controlled atmosphere (37aC, 5% CO2) in high glucose DMEM (Vero and L929) or MEM (Calu-3) supplemented with 7% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin.	Vero E6 suggested: None L929 suggested: ECACC Cat# 86032004, RRID:CVCL_4238) Vero suggested: None
   The levels of IL-6 (cat. No. DY206), IL-8 (cat. No. DY208) and TNF-α (cat. No. DY210) were quantified in the supernatants from uninfected and SARS-CoV-2-infected Calu-3 cells by ELISA (R&D Systems), following manufacturer’s instructions, and results are expressed as n-fold relative to uninfected cells.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Wild-type C57BL/6 (Central Animal House of the UFMG) and TNF receptor Knockout mice (TNFRp55-/-, Jackson Laboratories, stock No. 002818) were housed in individually ventilated cages placed in an animal care facility at 24 ± 2 °C on a 12-h light/ 12-h dark cycle, receiving ad libitum access to water and food.	C57BL/6 suggested: None
   On the fourth-day post infection, mice were euthanized, the dataloggers were collected and the body temperature readings of each animal were downloaded and analyzed (SubCue software, Calgary, AB, Canada).	AB suggested: RRID:BDSC_203)
   Software and Algorithms
   Sentences	Resources
   The acquisition was carried out in a BD FACSCanto II cell analyzer and analyzed using FlowJo software (Tree Star, Ashland, OR)	FlowJo suggested: (FlowJo, RRID:SCR_008520)

   ---

   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: We detected the following sentences addressing limitations in the study:

   As well as other animal models already proposed for COVID-19 study, the MHV-3 model also has its limitations. One of them is the difference in the host cell receptor used for viral entry. MHV-3 uses the CEACAM-1 receptor (32) whereas SARS-CoV-2 uses ACE2 (33), which may preclude studies of viral entry or drugs that act on this stage of the replication cycle. Another important limitation is the strong viral tropism towards liver cells and the impairment of the function of this organ in late time of infection. However, our results demonstrating significant lung damage and impairment of pulmonary functions following the first days after infection make our model quite useful to study coronavirus-associated lung inflammation and injury.

   ---

   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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 35 and 37. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2021.05.28.446200v1 on bioRxiv

   May 29, 2021