Journal
NATURE PROTOCOLS
Volume 10, Issue 12, Pages 1939-1947Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.121
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Funding
- US National Institutes of Health (NIH) [R01 1R01AI116892]
- University of Florida Opportunity Fund [00093472]
- NIH [R01 AI080611, R21 AI103961]
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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 RTRT-qPCR, requires similar to 6 h.
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