Effect of Short Time of SARS-CoV-2 Infection in Caco-2 Cells

Abstract

Coronavirus disease 19 (COVID-19) clinical manifestations include the involvement of the gastrointestinal tract, affecting around 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children. In the present work, the consequence of a short time of viral absorption (5, 15, 30 and 60 min) was tested on the Caco-2 intestinal epithelial cell line. Our findings show that Caco-2 cells are highly permissive to SARS-CoV-2 infection, even after 5 min of viral inoculation at a multiplicity of infection of 0.1. No cytopathic effect was evident during the subsequent 7 days of monitoring; nevertheless, the immunofluorescence staining for the viral nucleocapsid confirmed the presence of intracellular SARS-CoV-2. Our findings highlight the very short time during which SARS-CoV-2 is able to infect these cells in vitro.

Keywords: Caco-2 cell line; SARS-CoV-2; short time of infection; viral absorption.

Figure 1

The RNA viral load (expressed as Log10 RNA viral copies/mL) at 0, 3, 5 and 7 day post infection at the different MOI (0.1, 0.01, 0.001, 0.0001) and time of absorption ((A) 60 min; (B) 30 min; (C) 15 min; (D) 5 min). Results from Kruskal–Wallis test are displayed. ** p < 0.01, * p < 0.05.

Figure 2

The intracellular RNA viral load. (A) Intracellular viral load for N gene (expressed as Log10 RNA viral copies) at 7 days post infection at the different MOI (0.1, 0.01, 0.001, and 0.0001) and time of absorption (60, 30, 15, and 5 min). (B) Intracellular ratio of subgenomic E RNA viral copies (Log₁₀)/genomic E RNA viral copies (Log₁₀).

Figure 3

Viability of the cells infected with different virus concentrations (MOI 0.1, 0.01, 0.001, and 0.0001) and different timings of viral absorption (60, 30, 15, and 5 min) at 7 days post infection. The results are presented as percentage with respect to the cells not treated (indicated as NT in black).

Figure 4

Immunofluorescence staining for nucleocapsid SARS-CoV-2 protein with the experimental protocol employing 60 min of viral absorption (60× magnification, scale bar 30 micron). The fluorescence for nucleus (blue), F-actin cytoskeleton (red), nucleocapsid protein of SARS-CoV-2 (green) and the merge of the three channels are displayed. (A–D) MOI 0.1; (E–H) MOI 0.01; (I–L) MOI 0.001; (M–P) MOI 0.0001.

Figure 5

Immunofluorescence staining for nucleocapsid SARS-CoV-2 protein with the experimental protocol employing 30 min of viral absorption (60× magnification, scale bar 30 micron). The fluorescence for nucleus (blue), F-actin cytoskeleton (red), nucleocapsid protein of SARS-CoV-2 (green) and the merge of the three channels are displayed. (A–D) MOI 0.1; (E–H) MOI 0.01; (I–L) MOI 0.001; (M–P) MOI 0.0001.

Figure 6

Immunofluorescence staining for nucleocapsid SARS-CoV-2 protein with the experimental protocol employing 15 min of viral absorption (60× magnification, scale bar 30 micron). The fluorescence for nucleus (blue), F-actin cytoskeleton (red), nucleocapsid protein of SARS-CoV-2 (green) and the merge of the three channels are displayed. (A–D) MOI 0.1; (E–H) MOI 0.01; (I–L) MOI 0.001; (M–P) MOI 0.0001.

Figure 7

Immunofluorescence staining for nucleocapsid SARS-CoV-2 protein with the experimental protocol employing 5 min of viral absorption (60× magnification, scale bar 30 micron). The fluorescence for nucleus (blue), F-actin cytoskeleton (red), nucleocapsid protein of SARS-CoV-2 (green) and the merge of the three channels are displayed. (A–D) MOI 0.1; (E–H) MOI 0.01; (I–L) MOI 0.001; (M–P) MOI 0.0001.

Figure 8

Immunofluorescence staining for nucleocapsid SARS-CoV-2 protein cells not treated (60× magnification, scale bar 30 micron). The fluorescence for nucleus (blue, (A)), F-actin cytoskeleton (red, (B)), nucleocapsid protein of SARS-CoV-2 (green, (C)) and the merge of the three channels are displayed (D).