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  • Protocol Extension
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Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors


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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Fig. 1: HuNoV GII.P4-GII.4 replicates in WT AB, nacre and PTU-treated zebrafish larvae.
Fig. 2: HuNoV replication in WT AB zebrafish larvae is reproducible across labs.
Fig. 3: Microinjection setup in a biosafety cabinet in a BSL-2 facility.
Fig. 4: Unfertilized and non-properly developed zebrafish embryos.
Fig. 5: Correct orientation of zebrafish larvae for yolk injection.
Fig. 6: HuNoV GII.P7-GII.6 replication at 3 dpi at different temperatures.
Fig. 7: Development of edema or postural aberrations in zebrafish larvae.
Fig. 8: HuNoV GII.P7-GII.6 replication can be detected in a single zebrafish larva.
Fig. 9: Distribution of rhodamine B after an injection in the pericardial cavity of zebrafish larvae.

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Data availability

The data presented in Figs. 1, 2, 4, 6, 8 and 9 were generated for this protocol and are available from the corresponding author upon reasonable request.


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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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Joana Rocha-Pereira.

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The authors declare no competing interests.

Additional information

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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Related links

Key references using this protocol

Van Dycke, J. et al. PLoS Pathog. 15, e1008009 (2019):

Takaki, K. et al. Nat. Prot. 8, 1114–1124 (2013):

This protocol is an extension to: Nat. Protoc. 8, 1114–1124 (2013):

Supplementary information

Reporting Summary

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).

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