Exploring zebrafish larvae as a COVID-19 model: probable SARS-COV-2 replication in the swim bladder
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Abstract
Animal models are essential to understand COVID-19 pathophysiology and for pre-clinical 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, bloodstream, or yolk, did not result in detectable SARS-CoV-2 replication in wild-type larvae. However, when the virus was inoculated in the swim bladder, a modest increase in viral RNA was observed after 24 hours, suggesting a successful infection in some animals. This was confirmed by immunohistochemistry, with cells positive for SARS-CoV-2 nucleoprotein observed in the swim bladder. 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. 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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