Vero Cells as a Mammalian Cell Substrate for Human Norovirus

Figure 1. (A) GII.4 HuNoV detection by qRT-PCR. qRT-PCR analysis of RNA extracted from HuNoV-infected Vero cells. Data represent n = 3 + SEM. * p < 0.05. (B) Vero cells do not express detectable H-antigen. An indirect ELISA was used to detect the H-antigen on the cell surface of Vero cells and Caco-2 cells. No signal was observed for secondary only antibody controls (2°). Data represent n = 4 (Vero cells H-antigen ELISA) or n = 1 (Caco-2 cell H-antigen ELISA) + SEM. ** p < 0.01. (C) Vero cells express CD300ld/CD300lf. Anti-CD300lf (ectodomain) indirect ELISA of Vero cells following 24-h treatment ± 8.8 BAEE of trypsin. No signal was observed for secondary only antibody controls (2°). Data represent n = 3 + SEM. **** p < 0.0001. (D) CD300ld/CD300lf expression on Vero cells by immunofluorescence. A representative image is shown at 40× magnification from n = 3 biological replicates. CD300ld/CD300lf-associated fluorescence was not observed for secondary antibody only (2°) treated cells. The inset shows a CD300ld/CD300lf-expressing Vero cell.

Figure 2. (A) Vero cells are permissive for GII.4 Sydney HuNoV. HuNoV levels were examined over a 5-day time-course and peaked between 48–72 hpi by qRT-PCR. Data represent n = 3 + SEM. * p < 0.05 and **** p < 0.0001. (B) UV inhibits HuNoV replication. 1 h treatment with UV light inactivated HuNoV reducing replication at 72 hpi as measured by qRT-PCR. Data represent n = 3 + SEM. *** p < 0.001 and **** p < 0.0001. Vero cells are permissive for GII.3 (C) and GII.4 Yerseke (D) HuNoVs. qRT-PCR analysis over a 3-day time-course. Data represent n = 3 + SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Figure 3. Detection of GII.4 Sydney (A) and GII.3 (B) HuNoV VP1 by ELISA. OD (450 nm) were measured and fold-changes normalized to HuNoV negative stool input values were graphed. Data represent n = 3 (A) or n = 1 (B) + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (C) Increased VP1 and p48 staining frequency in HuNoV-infected Vero cells. Detection of single-positive (VP1+ or p48+) and double-positive (VP1+ p48+) Vero cell populations following HuNoV inoculation. Histograms generated following fluorescent bead compensation with the cell counts normalized to the mode. Data represent n = 1 from 5 biological replicates. (D) Visualization of GII.4 Sydney VP1 and p48 proteins by immunofluorescence assay. Representative image is shown at 40× magnification from n = 3 biological replicates. VP1- or p48-associated fluorescence were not observed for secondary antibody only (2°) treated cells. The inset shows a HuNoV-infected Vero cell.

Figure 4. Vero cells are permissive for exosome-associated GII.4 Sydney HuNoV. qRT-PCR analysis of a 3-day time-course. Data represent n = 3 + SEM. ** p < 0.01 and *** p < 0.001.

Figure 5. The knockdown of antiviral host gene expression increases GII.4 Sydney HuNoV replication. Treatment with pooled gene-specific siRNAs [50 nM] 48 h before infection resulted in substantial increases in HuNoV titers 72 hpi by qRT-PCR compared to non-targeting control (NTC) siRNA treated cells. Data represent n = 3 + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.