Skip to main content
SpringerLink
Log in

Zebrafish as a Model for the Study of Host-Virus Interactions

  • Protocol
  • First Online:
Innate Antiviral Immunity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1656))

Abstract

Zebrafish (Danio rerio) has become an increasingly important model for in vivo and in vitro studies on host-pathogen interaction, offering scientists with optical accessibility and genetic tractability, and a vertebrate-type immunity that can be separated into innate and adaptive ones. Although it is shown in previous studies that few species of viruses can naturally infect zebrafish, the spring viraemia of carp virus (SVCV), a rhabdovirus that causes contagious acute hemorrhagic viraemia in a variety of cyprinid fishes, can infect zebrafish by both injection and static immersion methods in laboratory conditions. In addition, SVCV can infect zebrafish fibroblast cell line (ZF4 cells), together with the Epithelioma papulosum cyprini (EPC) cell line (EPC cells), a common cell line used widely in fish disease research. The infection and propagation of SVCV in zebrafish and especially in these cell lines can be employed conveniently in laboratory for functional assays of zebrafish genes. The zebrafish, ZF4 and EPC cell, and SVCV can serve as a simple and efficient model system in understanding host-virus interactions. In the present chapter, we provide detailed protocols for the host-virus interaction analysis based on zebrafish embryos, ZF4/EPC cells, and SVCV, including infection methods of zebrafish embryos and cell lines, analyses of immune responses by quantitative PCR (qPCR) and RNA sequencing (RNA-Seq), antiviral assays based on ZF4 and EPC cells, and the analysis of host-virus interaction using luciferase assays. These protocols should provide efficient and typical means to address host-virus interactions in a more general biological sense.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

Similar content being viewed by others

References

  1. Lam SH, Chua HL, Gong Z et al (2004) Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study. Dev Comp Immunol 28:9–28

    Article  CAS  Google Scholar 

  2. Tobin DM, May RC, Wheeler RT (2012) Zebrafish: a see-through host and a fluorescent toolbox to probe host-pathogen interaction. PLoS Pathog 8:e1002349

    Article  CAS  Google Scholar 

  3. Levraud JP, Palha N, Langevin C et al (2014) Through the looking glass: witnessing host-virus interplay in zebrafish. Trends Microbiol 22:490–497

    Article  CAS  Google Scholar 

  4. Howe K, Clark MD, Torroja CF et al (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496:498–503

    Article  CAS  Google Scholar 

  5. Nasevicius A, Ekker SC (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26:216–220

    Article  CAS  Google Scholar 

  6. Hogan BM, Verkade H, Lieschke GJ et al (2008) Manipulation of gene expression during zebrafish embryonic development using transient approaches. Methods Mol Biol 469:273–300

    Article  CAS  Google Scholar 

  7. Bill BR, Petzold AM, Clark KJ et al (2009) A primer for morpholino use in zebrafish. Zebrafish 6:69–77

    Article  CAS  Google Scholar 

  8. Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785

    Article  CAS  Google Scholar 

  9. Wood AJ, Lo TW, Zeitler B et al (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333:307

    Article  CAS  Google Scholar 

  10. Cade L, Reyon D, Hwang WY et al (2012) Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Res 40:8001–8010

    Article  CAS  Google Scholar 

  11. Hwang WY, Fu Y, Reyon D et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229

    Article  CAS  Google Scholar 

  12. De Santis F, Di Donato V, Del Bene F (2016) Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology. Methods Cell Biol 135:171–188

    Article  Google Scholar 

  13. Crane M, Hyatt A (2011) Viruses of fish: an overview of significant pathogens. Virus 3:2025–2046

    Article  Google Scholar 

  14. Crim MJ, Riley LK (2012) Viral diseases in zebrafish: what is known and unknown. ILAR J 53:135–143

    Article  CAS  Google Scholar 

  15. Wang L, Wang L, Zhang HX et al (2006) In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus. J Interf Cytokine Res 26:256–259

    Article  Google Scholar 

  16. Ludwig M, Palha N, Torhy C et al (2011) Whole-body analysis of a viral infection: vascular endothelium is a primary target of infectious hematopoietic necrosis virus in zebrafish larvae. PLoS Pathog 7:e1001269

    Article  CAS  Google Scholar 

  17. LaPatra SE, Barone L, Jones GR et al (2000) Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish. Blood Cells Mol Dis 26:445–452

    Article  CAS  Google Scholar 

  18. Xu X, Zhang L, Weng S et al (2008) A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection. Virology 376:1–12

    Article  CAS  Google Scholar 

  19. Xiong XP, Dong CF, Xu X et al (2011) Proteomic analysis of zebrafish (Danio rerio) infected with infectious spleen and kidney necrosis virus. Dev Comp Immunol 35:431–440

    Article  CAS  Google Scholar 

  20. Lu MW, Chao YM, Guo TC et al (2008) The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model. Mol Immunol 45:1146–1152

    Article  CAS  Google Scholar 

  21. Phelan PE, Mellon MT, Kim CH (2005) Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Mol Immunol 42:1057–1071

    Article  CAS  Google Scholar 

  22. Phelan PE, Pressley ME, Witten PE et al (2005) Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio). J Virol 79:1842–1852

    Article  CAS  Google Scholar 

  23. Novoa B, Romero A, Mulero V et al (2006) Zebrafish (Danio rerio) as a model for the study of vaccination against viral haemorrhagic septicemia virus (VHSV). Vaccine 24:5806–5816

    Article  CAS  Google Scholar 

  24. Encinas P, Rodriguez-Milla MA, Novoa B et al (2010) Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach. BMC Genomics 11:518

    Article  Google Scholar 

  25. Lopez-Munoz A, Roca FJ, Sepulcre MP et al (2010) Zebrafish larvae are unable to mount a protective antiviral response against waterborne infection by spring viremia of carp virus. Dev Comp Immunol 34:546–552

    Article  CAS  Google Scholar 

  26. Lopez-Munoz A, Roca FJ, Meseguer J et al (2009) New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN-dependent genes and display powerful antiviral activities. J Immunol 182:3440–3449

    Article  CAS  Google Scholar 

  27. Zou PF, Chang MX, Xue NN et al (2014) Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant. Immunology 141:192–202

    Article  CAS  Google Scholar 

  28. Zou PF, Chang MX, Li Y et al (2015) Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish. Fish Shellfish Immunol 43:13–24

    Article  CAS  Google Scholar 

  29. Zou PF, Chang MX, Li Y et al (2016) NOD2 in zebrafish functions in antibacterial and also antiviral responses via NF-kappaB, and also MDA5, RIG-I and MAVS. Fish Shellfish Immunol 55:173–185

    Article  CAS  Google Scholar 

  30. Levraud JP, Boudinot P, Colin I et al (2007) Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system. J Immunol 178:4385–4394

    Article  CAS  Google Scholar 

  31. Burgos JS, Ripoll-Gomez J, Alfaro JM et al (2008) Zebrafish as a new model for herpes simplex virus type 1 infection. Zebrafish 5:323–333

    Article  CAS  Google Scholar 

  32. Antoine TE, Jones KS, Dale RM et al (2014) Zebrafish: modeling for herpes simplex virus infections. Zebrafish 11:17–25

    Article  CAS  Google Scholar 

  33. Ding CB, Zhao Y, Zhang JP et al (2015) A zebrafish model for subgenomic hepatitis C virus replication. Int J Mol Med 35:791–797

    Article  CAS  Google Scholar 

  34. Palha N, Guivel-Benhassine F, Briolat V et al (2013) Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish. PLoS Pathog 9:e1003619

    Article  CAS  Google Scholar 

  35. Gabor KA, Goody MF, Mowel WK et al (2014) Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment. Dis Model Mech 7:1227–1237

    Article  CAS  Google Scholar 

  36. He S, Salas-Vidal E, Rueb S et al (2006) Genetic and transcriptome characterization of model zebrafish cell lines. Zebrafish 3:441–453

    Article  CAS  Google Scholar 

  37. LF L, Li S, XB L et al (2016) Spring viremia of varp virus N protein suppresses fish IFNphi1 production by targeting the mitochondrial antiviral signaling protein. J Immunol 196:3744–3753

    Article  Google Scholar 

  38. Biacchesi S, LeBerre M, Lamoureux A et al (2009) Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses. J Virol 83:7815–7827

    Article  CAS  Google Scholar 

  39. Westerfield M, Streisinger G (1989) The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio). University of Oregon Press, Oregon

    Google Scholar 

  40. Chang MX, Collet B, Nie P et al (2011) Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in rainbow trout (Oncorhynchus mykiss). J Virol 85:8403–8512

    Article  CAS  Google Scholar 

  41. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by grants (31402273, 31320103913) from National Natural Science Foundation of China. We thank Dr. Shao Chen Pang in the Institute of Environment and Health, Jianghan University, Wuhan, China for providing a picture in Fig. 2.

Author information

Authors and Affiliations

  1. College of Fisheries, Jimei University, 43 Yindou Road, Xiamen, Fujian Province, 361021, China

    Peng Fei Zou

  2. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China

    Pin Nie

Authors
  1. Peng Fei Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pin Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pin Nie .

Editor information

Editors and Affiliations

  1. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada

    Karen Mossman

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Zou, P.F., Nie, P. (2017). Zebrafish as a Model for the Study of Host-Virus Interactions. In: Mossman, K. (eds) Innate Antiviral Immunity. Methods in Molecular Biology, vol 1656. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7237-1_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7237-1_2

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7236-4

  • Online ISBN: 978-1-4939-7237-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation