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

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ENTERIC VIRUSES REPLICATE IN SALIVARY GLANDS AND INFECT THROUGH SALIVA Article 29 June 2022 NOROVIRUS REPLICATION, HOST INTERACTIONS AND VACCINE ADVANCES Article 17 January 2025 MURINE

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(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 * Department of Molecular Genetics and Microbiology, University of Florida, Gainesville,

Florida, USA Melissa K Jones, Katrina R Grau, Scott A Tibbetts & Stephanie M Karst * Centers for Disease Control and Prevention, Atlanta, Georgia, USA Veronica Costantini & Jan Vinjé

* Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA Abimbola O Kolawole & Christiane E Wobus * Department of Viroscience; Erasmus Medical

Center, Miranda de Graaf & Marion Koopmans * Erasmus Medical Center, Rotterdam, The Netherlands Miranda de Graaf & Marion Koopmans * Department of Infectious Diseases, St. Jude

Children's Research Hospital, Memphis, Tennessee, USA Pamela Freiden & Stacey Schultz-Cherry * Department of Oral Biology, College of Dentistry, University of Florida, Gainesville,

Florida, USA Christina L Graves & Shannon M Wallet Authors * Melissa K Jones View author publications You can also search for this author inPubMed Google Scholar * Katrina R Grau View

author publications You can also search for this author inPubMed Google Scholar * Veronica Costantini View author publications You can also search for this author inPubMed Google Scholar *

Abimbola O Kolawole View author publications You can also search for this author inPubMed Google Scholar * Miranda de Graaf View author publications You can also search for this author

inPubMed Google Scholar * Pamela Freiden View author publications You can also search for this author inPubMed Google Scholar * Christina L Graves View author publications You can also

search for this author inPubMed Google Scholar * Marion Koopmans View author publications You can also search for this author inPubMed Google Scholar * Shannon M Wallet View author

publications You can also search for this author inPubMed Google Scholar * Scott A Tibbetts View author publications You can also search for this author inPubMed Google Scholar * Stacey

Schultz-Cherry View author publications You can also search for this author inPubMed Google Scholar * Christiane E Wobus View author publications You can also search for this author inPubMed

 Google Scholar * Jan Vinjé View author publications You can also search for this author inPubMed Google Scholar * Stephanie M Karst View author publications You can also search for this

author inPubMed Google Scholar 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 CITE THIS ARTICLE Jones, M., Grau, K., Costantini, V. _et al._ Human norovirus culture in B cells. _Nat Protoc_ 10, 1939–1947 (2015).

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