Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

Figures

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  FIGURE 1. Susceptibility of various cell lines to HuSaV infection. (A) Changes in HuSaV RNA copy numbers in the culture supernatants of HuTu80 and HEK293T cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~4 × 107 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean value of the HuSaV RNA copy numbers; error bars denote the geometric standard deviation (SD). This experiment was performed once with five technical replicates. (B) Changes in HuSaV RNA copy numbers in the culture supernatants of Caco‐2, HCT15, HCT116, Caco‐2/Cas9, and C2BBe1 cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~2 × 106 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars denote the geometric SD. This experiment was performed once with five technical replicates. (C) Immunofluorescence staining of the viral protein VP1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells at 3 dpi with a HuSaV GI.1 (AH20)‐positive stool suspension; upper panels: 4× objective lens, lower panels: 40× objective lens. (D) Flow cytometry…

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  FIGURE 2. Serial passage of HuSaV GI.1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells. HuSaV RNA copy numbers in the culture supernatants at 0 or 1 dpi (immediately after medium replacement) and 7 dpi for each passage, up to P + 5, in (A) HuTu80, (B) HEK293T, (C) Caco‐2, and (D) Caco‐2/Cas9 cells.

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  FIGURE 3. Effect of different bile acids on HuSaV replication efficiency in Caco‐2/Cas9 cells. Caco‐2/Cas9 cells were inoculated with HuSaV GI.1 (AH20)‐positive stool suspension (approximately 4 × 107 copies/well in 96‐well plates) and incubated at 37°C for 3 h. HuSaV RNA copy numbers in the culture supernatants were measured at 0 dpi (immediately after medium replacement) and 7 dpi with different bile acids in the culture media. Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars represent the geometric SD. This experiment was performed once with five technical replicates. GCA, Sodium glycocholate; GCDCA, sodium glycochenodeoxycholate.

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  FIGURE 4. Purification of HuSaV GI.1 from infected Caco‐2/Cas9 cells with CsCl density‐gradient ultracentrifugation. (A) HuSaV RNA copy numbers and density profiles across fractions from culture supernatants and cell lysates. Fractions marked with circles on the x‐axis were selected for subsequent electron microscopy western blotting analyses. (B) Electron microscopy of fractions 1, 2, 9, and 10 from the culture supernatants and fractions 9, 10, and 11 from the cell lysates. (C) Western blotting detection of the capsid protein VP1. The 60 kDa protein band, indicated by an arrow, corresponds to VP1. (D) Immunofluorescence staining for VP1 in Caco‐2/Cas9 cells at 3 dpi following re‐infection with fractions 1 and 10 from the culture supernatants and cell lysates.

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  FIGURE 5. Immunofluorescence and flow cytometry analysis of highly HuSaV‐susceptible Caco‐2MC cells. (A) Immunofluorescence staining at 4 dpi with HuSaV GI.1 (AH20) on cloned cells derived from Caco‐2/Cas9 cells; (left) clone M (Caco‐2M), (right) clone P (Caco‐2P); blue: Nuclei (Hoechst). (B) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2M, Caco‐2MC, and Caco‐2ME cells; blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected). (C) Immunofluorescence staining of Caco‐2MC cells at 4 dpi following infection with HuSaV GI.1 (AH20). (D) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2PG cells.

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  FIGURE 6. Time‐course analysis of RNA copy numbers and TCID50 in HuSaV‐infected Caco‐2MC cells. (A) Time‐course changes in RNA copy numbers in culture supernatants and cell lysates from 0 to 7 dpi. Circular dots indicate the geometric mean values of the HuSaV RNA copy numbers, and error bars represent geometric SD. This experiment was performed once with three technical replicates. (B) Time‐course changes in TCID50 values in supernatants and cell lysates from 0 to 7 dpi. Each dot represents individual data points; bars indicate the geometric mean TCID50 values; error bars represent geometric SD. This experiment was performed once with three technical replicates. (C) Phase‐contrast microscopy images of Caco‐2MC cells from 0 to 7 dpi; (top) HuSaV GI.1‐infected Caco‐2MC cells, (bottom) uninfected control cells.

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  FIGURE 7. Effect of different bile acids on HuSaV replication efficiency in Caco‐2MC cells. Caco‐2MC cells were inoculated with HuSaV GI.1 (M13‐18) stock at an MOI of 0.01 and incubated at 37°C for 3 h. HuSaV RNA copy numbers in the cell lysates were measured at 0 dpi (after 3 h incubation) and 5 dpi with different bile acids in the culture media. Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars represent the geometric SD. This experiment was performed three times with five technical replicates. GCA, sodium glycocholate; GCDCA, sodium glycochenodeoxycholate.

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  FIGURE 8. Serial passaging of multiple HuSaV genotypes in Caco‐2MC cells. The bar graphs show the HuSaV RNA copy numbers in the cell lysates at 0 and 5 dpi for each passage.

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  FIGURE 9. HuSaV neutralization assay using Caco‐2MC cells. HuSaV RNA copy numbers in the cell lysate at 5 dpi. Panel (A) shows results using anti‐rabbit HuSaV VLP GI.1 VP1 hyperimmune serum, while panel (B) shows results with non‐immunized rabbit serum. Each dot represents an individual data point, and curves were fitted using nonlinear regression in GraphPad Prism 8. This experiment was performed once with four technical replicates.