Research Article

Replication of human noroviruses in stem cell–derived human enteroids

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Science  23 Sep 2016:
Vol. 353, Issue 6306, pp. 1387-1393
DOI: 10.1126/science.aaf5211
  • Fig. 1 Replication of GII.4 variants in human intestinal enteroids (HIEs).

    (A) Jejunal HIE monolayers were inoculated with 9 × 105 genome equivalents of the indicated HuNoV GII.4 stool filtrates. RNA was extracted from cells and medium, and viral genome equivalents were 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 SD. Each experiment was performed two or more times, with three technical replicates in each experiment. In (B) to (E), monolayers inoculated with 9 × 107 genome equivalents of GII.4/2012-1 are shown; in (F), monolayers inoculated with 9 × 105 genome equivalents of GII.4/2012-1 are shown. (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 (4′,6-diamidino-2-phenylindole) 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 nonspecific band. (F) Kinetics of HuNoV yield at the indicated time points. (G) Passaging of GII.4/2009 HuNoV in jejunal HIEs. In (F) and (G), viral genome equivalents were quantified by RT-qPCR as in (A).

  • Fig. 2 Bile is required for GII.3 HuNoV replication and affects the cells.

    (A to C) Jejunal HIE monolayers were pretreated with the indicated concentrations of human bile for 2 days and then inoculated with GII.3 stool filtrate [(A) and (C), 4.3 × 105 genome equivalents; (B), 4.3 × 107 genome equivalents] and incubated with the same bile concentrations as used for pretreatment (see supplementary materials). (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) Determination of effect of bile treatment on virus or cells. 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% before infection of HIE monolayers not pretreated with bile; cells were either not treated or treated with 5% human bile for 2 days before 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). (F) HIE monolayers treated with bile as indicated in (E) were infected with 4.3 × 105 genome equivalents of GII.3 stool filtrate. For (A), (C), (D), and (F), genome equivalents were determined as indicated in Fig. 1. Error bars denote SD. *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, and GII.4/2012-1, 9 × 105 genome equivalents; GII.4/2006b-3, 5.5 × 105 genome equivalents; 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 SD.

  • 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 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 J2 HIE is included as control to show replication of GII.4 at 6 dpi. In (A) to (C), genome equivalents were determined as indicated in Fig. 1. Error bars denote SD.

  • Fig. 5 Inactivation of GII.4 and GII.3 HuNoV infectivity by γ irradiation and heat treatment.

    (A) GII.4/2012-1 and GII.3 HuNoVs were γ-irradiated or incubated at room temperature overnight. (B and C) GII.4/2012-1 (9 × 105 genome equivalents) (B) or GII.3 (4.3 × 105 genome equivalents) (C) were heat-inactivated at 60°C for the indicated time points or incubated at room temperature for 0 and 60 min. Jejunal HIEs were inoculated with each sample. Genome equivalents were determined as shown in Fig. 1. Error bars denote SD.

  • Table 1 Comparison of BT50 and 50% neutralization titers.

    BT50 denotes a serum titer that blocks the interaction of HuNoV VLPs with H type 1 and H type 3 and porcine gastric mucin glycans by 50%; 50% neutralization denotes a serum titer that reduces the infectivity of indicated HuNoV strains by 50%.

    SerumGII.3GII.4
    BT5050% neutralizationBT5050% neutralization
    1187990671105,000
    27083553214

Supplementary Materials

  • Replication of human noroviruses in stem cellâ€"derived human enteroids

    Khalil Ettayebi, Sue E. Crawford, Kosuke Murakami, James R. Broughman, Umesh Karandikar, Victoria R. Tenge, Frederick H. Neill, Sarah E. Blutt, Xi-Lei Zeng, Lin Qu, Baijun Kou, Antone R. Opekun, Douglas Burrin, David Y. Graham, Sasirekha Ramani, Robert L. Atmar, Mary K. Estes

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Table S1
    • References

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