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Analysis of resistance and tolerance to virus infection in Drosophila

Nature Protocols volume 10, pages 1084–1097 (2015)Cite this article

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Abstract

Host defense to virus infection involves both resistance mechanisms that reduce viral burden and tolerance mechanisms that limit detrimental effects of infection. The fruit fly, Drosophila melanogaster, has emerged as a model for identifying and characterizing the genetic basis of resistance and tolerance. This protocol describes how to analyze host responses to virus infection in Drosophila, and it covers the preparation of virus stocks, experimental inoculation of flies and assessment of host survival and virus production, which are indicative of resistance or tolerance. It also provides guidance on how to account for recently identified confounding factors, including natural genetic variation in the pastrel locus and contamination of fly stocks with persistent viruses and the symbiotic bacterium Wolbachia. Our protocol aims to be accessible to newcomers to the field and, although optimized for virus research using Drosophila, some of the techniques could be adapted to other host organisms and/or other microbial pathogens. Preparation of fly stocks requires ∼1 month, virus stock preparation requires 17–20 d, virus injection and survival assays require 10–15 d and virus titration requires 14 d.

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Figure 1: Overview of the experimental workflow.
Figure 2: Structure of the pastrel locus and location of SNPs.
Figure 3: Parameters that affect mortality in survival assays.
Figure 4: Practical setup for bleaching of embryos.
Figure 5: Intra-thoracic and intra-abdominal injection sites.

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Acknowledgements

We thank members of the van Rij laboratory for helpful discussions, and P. Miesen for critical reading of the manuscript. For providing fly stocks, we thank the Bloomington Stock Center. This work is supported by a PhD fellowship from the Radboud Institute for Molecular Life Sciences. Research in the laboratory is funded by an Excellent Chemisch Onderzoek (ECHO) project grant (grant number 711.013.001) and the Open Program of the Division for Earth and Life Sciences (grant number 821.02.028) from the Netherlands organization for scientific research (NWO) and a European Research Council consolidator grant under the European Union's Seventh Framework Programme (ERC grant number 615680).

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Authors and Affiliations

  1. Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands

    Sarah H Merkling & Ronald P van Rij

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  1. Sarah H Merkling
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Contributions

S.H.M. performed the experiments; S.H.M. and R.P.v.R. conceived and designed the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Ronald P van Rij.

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The authors declare no competing financial interests.

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Merkling, S., van Rij, R. Analysis of resistance and tolerance to virus infection in Drosophila. Nat Protoc 10, 1084–1097 (2015). https://doi.org/10.1038/nprot.2015.071

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