Fig. 1.
Replication of GII.4 variants in human intestinal enteroids. Jejunal HIE monolayers were inoculated with (A) 9×105 genome equivalents of the indicated HuNoV GII.4 stool filtrates. RNA was extracted from cells and medium, and viral genome equivalents quantified by RT-qPCR. RNA at 1 hpi was collected after removal of virus inoculum and washing of cells twice to remove any unattached viruses. Each data bar represents the mean of three wells of inoculated HIEs. Error bars denote standard deviation. Each experiment was performed two or more times, with three technical replicates in each experiment. Panels (B-E) and (F) represent monolayers inoculated with 9×107 and 9×105 genome equivalents of GII.4/2012-1, respectively. (B) Expression of VP1 was detected in enterocytes (villin, red) in formalin-fixed, paraffin-embedded enteroid monolayer sections using antibody against GII.4/2012 VLPs (green). DAPI detects nuclei (blue). Scale bar = 25 µm. (C) Flow cytometry quantitation and immunofluorescent detection of infected cells. Scale bar = 100 µm. (D) Electron micrograph of HuNoV particles from the supernatant of infected HIEs. Scale bar = 50 nm. Inset: small particle. Scale bar = 25 nm. (E) Western blot detecting polyprotein processing and VP1 expression. Asterisk marks a non-specific band. (F) Kinetics of HuNoV yield at the indicated time points. (G) Passaging of GII.4/2009 HuNoV in jejunal HIEs. (F, G) Viral genome equivalents quantified by RT-qPCR as indicated for panel A.