Cited 143 times since 2015 (16.7 per year) source: EuropePMC Nature protocols, Volume 10, Issue 12, 29 5 2015, Pages 1939-1947 Human norovirus culture in B cells. Jones MK, Grau KR, Costantini V, Kolawole AO, de Graaf M, Freiden P, Graves CL, Koopmans M, Wallet SM, Tibbetts SA, Schultz-Cherry S, Wobus CE, Vinjé J, Karst SM
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.
