Replication of Human Sapovirus in Human-Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells

Figures

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  Figure 1. Requirement for bile during the in vitro replication of HuSaV. Monolayers of human iPSC-derived IECs were cultured with or without 0.03% porcine bile and then inoculated with 2 × 106 genome equivalents of the indicated HuSaV genotypes. Inoculation and sampling were performed as described in the Methods section. Viral genome RNA was extracted from supernatants sampled at 1 or 48 hpi, and the genome equivalents were quantified by RT-qPCR. Each value is representative of at least three independent experiments and is shown as the mean ± SD from four wells of supernatants from each culture group. The mean fold changes in viral genome between the two time points are indicated above each black bar (48 hpi).

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  Figure 2. Replication of HuSaVs in IECs derived from human iPSCs. Monolayers of human iPSC-derived IECs were inoculated with the indicated genome equivalents (A) or 2 × 107 genome equivalents (B,C) of the indicated HuSaV genotypes. Inoculation and sampling were performed as described in the Methods section. Viral genomic RNA was extracted from supernatants sampled at 1 or 48 h postinfection (hpi) (A,C) or the indicated times (B), and genome equivalents were then quantified by real-time PCR. Each value is representative of at least three independent experiments and is shown as the mean ± SD from four (A,C) or three (B) wells of supernatants from each culture group. The mean fold changes in the viral genome between the two time points are indicated above each black bar (48 hpi). Dashed lines represent the limit of detection.

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  Figure 3. Inactivation of HuSaVs by heat and acid-ethanol treatments. GI.2 and GII.1 HuSaVs were incubated at 85 °C for the indicated times (A) or were suspended with 3 volumes of the indicated disinfectants at room temperature for 5 min (B). Monolayers of IECs were inoculated with 2 × 107 (A), 5.3 × 106 (B, GI.2), or 2.5 × 107 (B, GII.1) genome equivalents of the indicated HuSaV genotypes. Viral genome RNA was extracted from both supernatants, and the genome equivalents were quantified by real-time PCR. Samples at 1 hpi were used as references. Each value is representative of three independent experiments and is shown as the mean ± SD from four wells of supernatants for each culture group. The mean fold changes in viral genome between the two time points are indicated above each black bar (48 hpi). EtOH, 70% ethanol; MgSO4, 0.05% magnesium sulfate; Citric A, 1% citric acid. * p < 0.05. Dashed lines represent the limit of detection.

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  Figure 4. Neutralization of GII.1 HuSaV by antiserum induced with the immunization of GII.1 HuSaV VLPs. First, 2 × 107 genome equivalents of GII.1 or GII.3 HuSaVs were incubated with 0.2 μL of serum prepared from non-immunized mice (N.I.) or anti GII.1 HuSaV antiserum at 37 °C for 90 min before inoculation into the prepared IECs. Viral genomic RNA was extracted from supernatants sampled at 1 or 72 hpi, and genome equivalents were then quantified by real-time PCR. Each value is representative of three independent experiments and is shown as the mean ± SD from six wells of supernatants from each culture group. The mean fold changes in viral genome between the two time points are indicated above each black bar (72 hpi). * p < 0.05. Dashed lines represent the limit of detection.