Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells

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1. Version published to 10.1128/jvi.00608-22

   Jul 27, 2022
2. 

   ScreenIT

   Jun 3, 2021

   SciScore for 10.1101/2021.05.31.446374: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Bat primary cells: M. myotis samples were collected in July 2020 from two bat colonies in Inca and Llucmajor on Mallorca (Balearic Islands, Spain) (agreement CEP 31/2020 delivered by the Ministry of the Environment and Territory, government of the Balearic Islands). Euthanasia Agents: The bat was anesthetized with isofluranum (Piramal Enterprises Ltd.) and euthanized by quick decapitation.
   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

   Antibodies
   Sentences	Resources
   Cells were incubated with goat pAB anti-hACE2-647 (1:100, FAB933R R&D Systems) and/or with …

   SciScore for 10.1101/2021.05.31.446374: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Bat primary cells: M. myotis samples were collected in July 2020 from two bat colonies in Inca and Llucmajor on Mallorca (Balearic Islands, Spain) (agreement CEP 31/2020 delivered by the Ministry of the Environment and Territory, government of the Balearic Islands). Euthanasia Agents: The bat was anesthetized with isofluranum (Piramal Enterprises Ltd.) and euthanized by quick decapitation.
   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

   Antibodies
   Sentences	Resources
   Cells were incubated with goat pAB anti-hACE2-647 (1:100, FAB933R R&D Systems) and/or with antibodies recognizing the spike protein of SARS-CoV (anti-S, 1:1000, GTX632604 Genetex) or anti-S mAb10 (1 μg/ml, a kind gift from Dr. Hugo Mouquet, Institut Pasteur, Paris, France) and subsequently with secondary antibodies anti-human AlexaFluor-647 (1:1000, A21455 Thermo), anti-mouse AlexaFluor-488 (1:1000, A28175 Thermo) or Dylight488 (1:100, SA5-10166 Thermo) for 30 min at 4°C.	anti-hACE2-647 suggested: None anti-S suggested: (Acris Antibodies GmbH Cat# AP09205DL5-N, RRID:AB_2035586) anti-human AlexaFluor-647 suggested: None
   Subsequently, cells were incubated with anti-goat Alexa488 (A-11055, Thermo Fisher Scientific) and anti-mouse Alexa555 (A21427, Thermo Fisher Scientific) secondary antibodies diluted 1:500 in AB buffer for 30 min at RT.	anti-goat suggested: (Thermo Fisher Scientific Cat# A-11055, RRID:AB_2534102) anti-mouse suggested: (Innovative Research Cat# A21427, RRID:AB_1500655) A21427 suggested: (Innovative Research Cat# A21427, RRID:AB_1500655)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: FLG-ID, FLG-R, FLN-ID, FLN-R and Tb1Lu cell lines (table 1) were maintained in equal volumes of Ham’s F12 and Iscove’s modified Dulbecco’s medium (IMDM, Gibco), supplemented with 10% FBS and 1% P/S (Gibco) in non-vented flasks.	Tb1Lu suggested: None
   Mm and Nn cell lines (table 1), as well as African green monkey Vero E6 cells (ATCC CRL-1586), human lung epithelial A549 cells (kind gift from Frédéric Tangy, Institut Pasteur, Paris) and human colorectal adenocarcinoma Caco TC7 cells (ATCC HTB-37), were maintained in DMEM (Gibco), supplemented with 10% FBS and 1% P/S in vented flasks.	A549 suggested: None
   The cleared supernatant was stored at −80°C and titrated on Vero E6 cells by using standard plaque assays to measure plaque-forming units per mL (PFU/mL).	Vero E6 suggested: RRID:CVCL_XD71)
   Recombinant DNA
   Sentences	Resources
   Primary cells were immortalized by transfection of pRSVAg1 plasmid expressing Simian Vacuolating Virus 40 large T antigen (SV40T) with lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, expanded and cryopreserved.	pRSVAg1 suggested: None
   All cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Bat and A549 cells were modified to stably express hACE2 using the pLenti6-hACE2 lentiviral transduction as described previously [43].	pLenti6-hACE2 suggested: None
   Genome equivalent concentrations were determined by extrapolation from a standard curve generated from serial dilutions of the pcDNA3.1-hACE2 plasmid (addgene, 145033) or plasmids encoding a fragment of the RNA-dependent RNA polymerase (RdRp)-IP4 of SARS-CoV-2 or a fragment of the ACE2 genome of each bat species.	pcDNA3.1-hACE2 suggested: RRID:Addgene_145033)
   PCR products were gel-purified (NucleoSpin gel and PCR clean-up kit, Macherey-Nagel) and cloned into pCR-Blunt II-TOPO vectors using the Zero Blunt TOPO PCR Cloning Kit (Thermo).	pCR-Blunt II-TOPO suggested: RRID:Addgene_29705)
   Software and Algorithms
   Sentences	Resources
   Cells were acquired on an Attune NxT Flow Cytometer (Thermo Fisher) and data analyzed with FlowJo software v10 (TriStar)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: Graphical representation and statistical analyses were performed using GraphPad Prism Version 9.0.2 software (GraphPad).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) 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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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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.
   • 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.

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

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3. 

   Version published to 10.1101/2021.05.31.446374v1 on bioRxiv

   May 31, 2021