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Human norovirus culture in B cells

Nature Protocols volume 10, pages 1939–1947 (2015)Cite this article

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Abstract

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

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Figure 1: Viral input levels inversely correlate with infection efficiency.
Figure 2: HuNoV infection of B cells is reproducible.
Figure 3: HuNoV replication in B cells is cell line–specific.
Figure 4: BJAB morphology.

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Acknowledgements

This work was funded by the US National Institutes of Health (NIH) R01 1R01AI116892 and the University of Florida Opportunity Fund 00093472 for S.M.K., and by NIH R01 AI080611 and R21 AI103961 for C.E.W. We would like to thank G. McFadden (University of Florida), R. Renne (University of Florida) and B. Chandran (Rosalind Franklin University of Medical School and Sciences) for generously providing cell lines used in these studies. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Names of specific vendors, manufacturers or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers or products by the CDC or the US Department of Health and Human Services.

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Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA

    Melissa K Jones, Katrina R Grau, Scott A Tibbetts & Stephanie M Karst

  2. Centers for Disease Control and Prevention, Atlanta, Georgia, USA

    Veronica Costantini & Jan Vinjé

  3. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA

    Abimbola O Kolawole & Christiane E Wobus

  4. Department of Viroscience; Erasmus Medical Center,

    Miranda de Graaf & Marion Koopmans

  5. Erasmus Medical Center, Rotterdam, The Netherlands

    Miranda de Graaf & Marion Koopmans

  6. Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Pamela Freiden & Stacey Schultz-Cherry

  7. Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA

    Christina L Graves & Shannon M Wallet

Authors
  1. Melissa K Jones
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Contributions

M.K.J., K.R.G., S.A.T. and S.M.K. developed and optimized direct and co-culture infections of BJAB cells, attachment assays, RNA extraction and qPCR parameters. C.L.G. and S.M.W. contributed to the development of the coculture infections. M.K.J. performed direct and co-culture infections. J.V. and V.C. provided GII.4-Sydney-positive stool samples and performed extensive numbers of infections in their CDC laboratory, investigating assay variables, and they also performed infections at the University of Florida. A.O.K. and C.E.W. performed infections at the University of Florida and the University of Michigan. P.F. and S.S.-C. performed direct infections at St. Jude Children's Research Hospital using stool samples provided by the CDC and collected at St. Jude's. M.d.G. and M.K. performed infections at the University of Florida and EMC. M.K.J., K.R.G. and S.M.K. jointly wrote the manuscript and prepared the figures. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Stephanie M Karst.

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Competing interests

A patent pertinent to this work has been filed (International application number PCT/US2015/030361, Methods and Compositions for Caliciviridae, M.K.J. and S.M.K. as inventors).

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Jones, M., Grau, K., Costantini, V. et al. Human norovirus culture in B cells. Nat Protoc 10, 1939–1947 (2015). https://doi.org/10.1038/nprot.2015.121

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