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Nat Protoc. 2015 Dec;10(12):1939-47. doi: 10.1038/nprot.2015.121. Epub 2015 Oct 29.

Human norovirus culture in B cells.

Author information

1
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.
2
Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
3
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
4
Department of Viroscience; Erasmus Medical Center, Rotterdam, The Netherlands.
5
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
6
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA.

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.

PMID:
26513671
PMCID:
PMC4689599
DOI:
10.1038/nprot.2015.121
[Indexed for MEDLINE]
Free PMC Article

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