Exploring Zebrafish Larvae as a COVID-19 Model: Probable Abortive SARS-CoV-2 Replication in the Swim Bladder
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Abstract
Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro . In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.
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SciScore for 10.1101/2021.04.08.439059: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization A randomized subset of larvae was then transferred to tubes and individually lysed in 320 µl of RLT buffer + 1% β-mercaptoethanol. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources To generate concentrated virus, Vero-E6 cells were infected with virus at an MOI of 0.01 PFU/cell in DMEM/2%FBS, and incubated for 72 h at 37°C, 5% CO2. Vero-E6suggested: NoneThe SINV-GFP virus corresponds to the SINV-eGFP/2A strain described in (Boucontet et al., 2018) and was used as a BHK cell supernatant at 2.107 … SciScore for 10.1101/2021.04.08.439059: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization A randomized subset of larvae was then transferred to tubes and individually lysed in 320 µl of RLT buffer + 1% β-mercaptoethanol. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources To generate concentrated virus, Vero-E6 cells were infected with virus at an MOI of 0.01 PFU/cell in DMEM/2%FBS, and incubated for 72 h at 37°C, 5% CO2. Vero-E6suggested: NoneThe SINV-GFP virus corresponds to the SINV-eGFP/2A strain described in (Boucontet et al., 2018) and was used as a BHK cell supernatant at 2.107 PFU/mL. BHKsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Briefly, 12.5µL of plasmid is mixed with 1.5µl of Custmart buffer and 1µl of I-SceI (New England Biolabs), and incubated at room temperature for 5 min before being put on ice until injection of 1 nL inside the cell of AB embryos at the one-cell stage. ABsuggested: RRID:BDSC_203)Software and Algorithms Sentences Resources Quantitation of sense or antisense viral N transcripts was performed by a Taqman probe assay, using the primer-probe mix from the 2019-nCoV RUO kit (IDT) with iTaq Universal Probes One-Step kit (Bio-Rad). iTaq Universal Probessuggested: NoneThey were further processed (superposition of channels, rotation, crop, and fluorescence intensity measure) using Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)Cell suspensions were acquired on an Attune NxT flow cytometer (ThermoFisher) with blue and yellow lasers, and data analyzed with FlowJo. FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistical analysis: Analysis were performed with GraphPad Prism. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
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.
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