Replication of human noroviruses in stem cell-derived human enteroids

Abstract

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

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.

Fig. 2

Bile is required for GII.3 HuNoV replication and affects the cells. Jejunal HIE monolayers were pretreated (A-C) with the indicated concentrations of human bile for 2 days and then inoculated with GII.3 stool filtrate [(A, C) 4.3×10⁵ or (B) 4.3×10⁷ genome equivalents] and incubated with the same bile concentrations as used for pretreatment (see supplementary methods). (B) VP1 was detected in methanol-fixed monolayers at 24 hpi using guinea pig anti-GII.3 VLP antiserum (green) and DAPI to detect nuclei (blue). Scale bar = 25 µm. (D) To determine if the effect of bile was on the virus or the cells, the virus was either not treated or treated with 5% human bile for 1 hour at 37°C, and then diluted to decrease the bile concentration to 0.025% prior to infection of HIE monolayers not pretreated with bile. Alternatively, cells were either not treated or treated with 5% human bile for 2 days prior to and during infection. Inoculations were performed with 4.3×10⁵ genome equivalents. (E) Schematic showing with black arrows when bile was added to HIEs for the experiment shown in (F). For A, C, D and F, genome equivalents were determined as indicated in Fig. 1. Error bars denote standard deviation. *, P< 0.05 comparing genome equivalents to 1 hpi.

Fig. 3

GII.4 variants and GII.3 HuNoVs replicate in HIEs generated from different intestinal segments. Duodenal (D1), jejunal (J2), and ileal (IL16) HIEs were treated with 1% sow bile for the GII.4 variants or 5% human bile for GII.3 for 48 hours, and then inoculated with the indicated HuNoVs (GII.4/2006b-2, GII.4/2009, GII.4/2012-1: 9×10⁵; GII.4/2006b-3: 5.5×10⁵; GII.3: 4.3×10⁵ genome equivalents) and cultured in the presence of bile. Genome equivalents were determined as indicated in Fig. 1. Error bars denote standard deviation.

Fig. 4

Replication of GII.4 strains but not GII.3 depends on HIE secretor status. (A) Secretor positive jejunal (J2, J3, J6 and J11) or (B) secretor negative jejunal (J4, J8 and J10) HIEs were inoculated with the indicated GII.4 or GII.3 HuNoVs (with the same amounts of genome equivalents as indicated in Fig. 3) in the presence of bile (1% sow bile for GII.4 variants or 5% human bile for GII.3) for 96 hours. At 96 hpi, GII.4 strains replicate in secretor positive HIEs but not secretor negative lines, while GII.3 replicates in all secretor positive HIEs and one secretor negative line (J8). (C) At 6 dpi, the GII.3 virus shows replication in an additional secretor negative HIE (J4) while no growth of GII.4/2012-1 virus is seen. A secretor positive J2 HIE is included as control to show replication of GII.4 at 6 dpi. (A–C) Genome equivalents were determined as indicated in Fig. 1. Error bars denote standard deviation.

Fig. 5

Inactivation of GII.4 and GII.3 HuNoV infectivity by gamma irradiation and heat treatment. (A) GII.4/2012-1 and GII.3 HuNoVs were gamma irradiated or incubated at room temperature overnight. (B) GII.4/2012-1 or (C) GII.3 (9×10⁵ and 4.3×10⁵ genome equivalents, respectively) were heat-inactivated at 60°C for the indicated time points or incubated at room temperature for 0 and 60 minutes. Jejunal HIEs were inoculated with each sample. Genome equivalents were determined as indicated in Fig. 1. Error bars denote standard deviation.