Human norovirus culture in B cells
Nature Protocols , Volume 10 - Issue 12 p. 1939- 1947
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.
|Organisation||Department of Virology|
Jones, M.K, Grau, K.R, Costantini, V, Kolawole, A.O, de Graaf, M.T, Freiden, P, … Karst, S.M. (2015). Human norovirus culture in B cells. Nature Protocols, 10(12), 1939–1947. doi:10.1038/nprot.2015.121