Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Human norovirus culture in B cells

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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Patel, M.M. Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg. Infect. Dis. 14, 1224–1231 (2008).

    Article  Google Scholar 

  2. Ahmed, S.M. et al. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect. Dis. 14, 725–730 (2014).

    Article  Google Scholar 

  3. Hall, A.J. et al. Norovirus disease in the United States. Emerg. Infect. Dis. 19, 1198–1205 (2013).

    Article  Google Scholar 

  4. Koo, H.L. et al. Noroviruses: the most common pediatric viral enteric pathogen at a large university hospital after introduction of rotavirus vaccination. J. Pediatr. Infect. Dis. Soc. 2, 57–60 (2013).

    Article  Google Scholar 

  5. Payne, D.C. et al. Norovirus and medically attended gastroenteritis in US children. N. Engl. J. Med. 368, 1121–1130 (2013).

    CAS  Article  Google Scholar 

  6. Koo, H.L., Ajami, N., Atmar, R.L. & DuPont, H.L. Noroviruses: the principal cause of foodborne disease worldwide. Discov. Med. 10, 61–70 (2010).

    PubMed  PubMed Central  Google Scholar 

  7. Duizer, E. et al. Laboratory efforts to cultivate noroviruses. J. Gen. Virol. 85, 79–87 (2004).

    CAS  Article  Google Scholar 

  8. Herbst-Kralovetz, M.M. et al. Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells. Emerg. Infect. Dis. 19, 431–438 (2013).

    CAS  Article  Google Scholar 

  9. Papafragkou, E., Hewitt, J., Park, G.W., Greening, G. & Vinje, J. Challenges of culturing human norovirus in a 3-D organoid cell culture model. PLoS ONE 8, e63485 (2013).

    CAS  Article  Google Scholar 

  10. Takanashi, S. et al. Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures. Arch. Virol. 159, 257–266 (2013).

    Article  Google Scholar 

  11. Marionneau, S. et al. Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals. Gastroenterology 122, 1967–1977 (2002).

    CAS  Article  Google Scholar 

  12. Gonzalez-Hernandez, M.B. et al. Murine norovirus transcytosis across an in vitro polarized murine intestinal epithelial monolayer is mediated by M-like cells. J. Virol. 87, 12685–12693.

  13. Tamura, M., Natori, K., Kobayashi, M., Miyamura, T. & Takeda, N. Interaction of recombinant Norwalk virus particles with the 105-kilodalton cellular binding protein, a candidate receptor molecule for virus attachment. J. Virol. 74, 11589–11597 (2000).

    CAS  Article  Google Scholar 

  14. White, L.J. et al. Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines. J. Virol. 70, 6589–6597 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Wobus, C.E. et al. Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2, e432 (2004).

    Article  Google Scholar 

  16. Ward, J.M. et al. Pathology of immunodeficient mice with naturally occurring murine norovirus infection. Toxicol. Pathol. 34, 708–715 (2006).

    Article  Google Scholar 

  17. Mumphrey, S.M. et al. Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses. J. Virol. 81, 3251–3263 (2007).

    CAS  Article  Google Scholar 

  18. Lay, M.K. et al. Norwalk virus does not replicate in human macrophages or dendritic cells derived from the peripheral blood of susceptible humans. Virology 406, 1–11 (2010).

    CAS  Article  Google Scholar 

  19. Basic, M. et al. Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10–deficient mice. Inflamm. Bowel Dis. 20, 431–443 (2014).

    Article  Google Scholar 

  20. Zhu, S. et al. Identification of immune and viral correlates of norovirus protective immunity through comparative study of intra-cluster norovirus strains. PLoS Pathog. 9, e1003592 (2013).

    CAS  Article  Google Scholar 

  21. Bok, K. et al. Chimpanzees as an animal model for human norovirus infection and vaccine development. Proc. Natl. Acad. Sci. USA 108, 325–330 (2011).

    CAS  Article  Google Scholar 

  22. Jones, M.K. et al. Enteric bacteria promote human and murine norovirus infection of B cells. Science 346, 755–759 (2014).

    CAS  Article  Google Scholar 

  23. Karst, S.M. Identification of a novel cellular target and a co-factor for norovirus infection: B cells and commensal bacteria. Gut Microbes 6, 266–71 (2015).

    CAS  Article  Google Scholar 

  24. Karst, S.M. & Wobus, C.E. A working model of how noroviruses infect the intestine. PLoS Pathog. 11, e1004626 (2015).

    CAS  Article  Google Scholar 

  25. Guix, S. et al. Norwalk virus RNA is infectious in mammalian cells. J. Virol. 81, 12238–12248 (2007).

    CAS  Article  Google Scholar 

  26. Lohmann, V. & Bartenschlager, R. On the history of hepatitis C virus cell culture systems. J. Med. Chem. 57, 1627–1642 (2013).

    Article  Google Scholar 

  27. Park, Y., Cho, Y.-H. & Ko, G. A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in both clinical and environmental specimens. Virus Genes 43, 192–200 (2011).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

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).

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.121

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing