Abstract
We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3–4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis.
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Acknowledgements
We very much appreciate the expert technical assistance and dedication of J. Rymenants, L. Bervoets and J. Maes. We thank the KU Leuven aquatic facility for breeding the zebrafish larvae. We thank the pediatrics department and the CEMOL Molecular Diagnostic department of the University Hospital of Leuven for the collaboration. J.V.D. and the research leading to these results has received funding from the Scientific Fund for Research of Flanders (FWO) as an SB doctoral fellow. A.C. is supported by internal funding from the KU Leuven.
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Contributions
Designed experiments: J.V.D., A.C., S.R., S.T., J.R.-P. Performed experiments: J.V.D., A.C., J.K. Analyzed data: J.V.D., A.C., J.K., S.T., J.R.-P. Supervised the experiments: A.N., P.d.W., S.T., J.N., J.R.-P. Wrote manuscript: J.V.D., A.C., S.T., J.R.-P. Graphs: J.V.D., A.C., J.K., S.T., J.R.-P. Figures and videos: A.C., J.V.D. All authors reviewed and edited the protocol.
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Peer review information Nature Protocols thanks Jean-Pieere Levraud and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Van Dycke, J. et al. PLoS Pathog. 15, e1008009 (2019): https://doi.org/10.1371/journal.ppat.1008009
Takaki, K. et al. Nat. Prot. 8, 1114–1124 (2013): https://doi.org/10.1038/nprot.2013.068
This protocol is an extension to: Nat. Protoc. 8, 1114–1124 (2013): https://doi.org/10.1038/nprot.2013.068
Supplementary information
Supplementary Video 1
Clipping of the microinjection needle
Supplementary Video 2
Correct size bolus
Supplementary Video 3
Microinjection in the yolk of 3 dpf zebrafish larvae
Supplementary Video 4
Dechorionation of 2 dpf zebrafish larvae
Supplementary Video 5
Microinjection in the pericardial cavity of 3 dpf zebrafish larvae
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Van Dycke, J., Cuvry, A., Knickmann, J. et al. Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors. Nat Protoc 16, 1830–1849 (2021). https://doi.org/10.1038/s41596-021-00499-0
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DOI: https://doi.org/10.1038/s41596-021-00499-0
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