Human norovirus culture in B cells

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.

Figure 1

Viral input levels inversely correlate with infection efficiency. BJAB cells were inoculated with increasing numbers of viral genome copies in unfiltered GII.4-Sydney HuNoV-positive stool, as indicated on the x axis. Viral genomes were enumerated at 0 and 3 d.p.i., and they are reported as the log of the fold increase in copies over time. Duplicate samples were tested per condition, and the entire experiment was repeated two times. Values from all replicates per condition were averaged. Error bars denote s.e.m.

Figure 2

HuNoV infection of B cells is reproducible. (a) Research personnel from the Centers for Disease Control and Prevention (CDC), Erasmus Medical Center (EMC) and the University of Michigan (UM) have successfully infected BJAB cells with GII.4-Sydney HuNoV when performing infections at the University of Florida (UF). Each bar represents the average fold-increase from duplicate or triplicate wells of a single experiment performed at UF. (b) Research personnel at UM have successfully infected BJAB cells with the same stock of GII.4-Sydney HuNoV at UM. Data are reported as the fold increase in genome copies from 0 to 3 d.p.i. at differing inoculation volumes, as indicated on the x axis. Quadruple samples were tested per condition and the entire experiment repeated three times. Values from all replicates per condition were averaged. (c) BJAB cells were cultured in medium containing FBS from three different sources, labeled A, B and C, and then inoculated with GII.4-Sydney HuNoV. Viral genomes were enumerated and the data are reported as fold increase between 0 and 3 d.p.i. Duplicate wells per condition were tested, and the entire experiment was repeated twice. Error bars denote s.e.m. in all panels.

Figure 3

HuNoV replication in B cells is cell line–specific. The Burkitt lymphoma B cell lines BJAB (EBV-negative), Raji (EBV-positive) and Namalwa (EBV-positive) and the myeloma line HuNS1 (EBV-negative) were inoculated with unfiltered GII.4-Sydney HuNoV-positive stool inoculum. Viral genome copy numbers were determined at 0 and 3 d.p.i. for each cell line. Duplicate samples were tested per condition, and the entire experiment was repeated two times. The data are reported as the log of the fold increase in copy numbers over time, and values from all replicates per condition were averaged. Error bars denote s.e.m.

Figure 4

BJAB morphology. BJAB cells at 4 d after being split 1:4 should have a smooth, round morphology and clumps consisting of >40 cells. Cells were imaged using a Leica DFC350 FX microscope and Leica Application Suite LAS AF AF6000 software.