Article Figures & Data
Figures
- FIG. 1.
ACE2 expression in kidney cell lines. Kidney cell lines derived from African green monkeys (Vero E6), human kidney PTEC, MC, and podocytes were analyzed for ACE2 expression by labeling the cell surface with an anti-ACE2 polyclonal Ab. The full histograms indicate cells testing positive for ACE2 expression, whereas the open histograms represent the staining with a control Ab (swine anti-goat phycoerythrin-conjugated secondary Ab). The number on the upper right indicates the percentage of ACE2+ cells.
- FIG. 2.
Kinetics of SARS-CoV replication in kidney cells. Vero E6 cells, PTEC, MC, and podocytes were infected with SARS-CoV HSR1 at an MOI of 0.1. Virus replication was measured by real-time PCR of the full genome (▴) in the supernatant of infected cells harvested every 24 h up to 4 days p.i. The quantification of subgenomic transcripts was carried out by real-time PCR on retrotranscribed cDNA obtained from infected cells every 24 h up to 4 days p.i. The nucleocapsid (N) log10 copy number (▪) was normalized by 50 ng of 18S RNA measured by real-time PCR. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log10/ml obtained in three independent experiments.
- FIG. 3.
Kinetics of SARS-CoV growth. The supernatants from infected PTEC (▪), MC (•), and Vero E6 (▴) cells, harvested every 24 h up to 4 days p.i., were tested in a Vero E6 plaque assay to determine the levels of infectious virus. Values represent the mean PFU ± standard deviation, expressed as log10/ml obtained in three independent experiments.
- FIG. 4.
Cell viability after SARS-CoV HSR1 infection of Vero E6 and human kidney cells. Cells were exposed to 4 × 105 PFU of SARS-CoV HSR1 and stained with Trypan blue dye after 72 h p.i. Photographs were taken using a digital camera connected to the light microscope at a magnification of ×40. CPE (dark) was evident in Vero E6 cells, whereas SARS-CoV-infected human kidney epithelial cells did not show decreased viability in comparison to uninfected control cell cultures.
- FIG. 5.
Persistent virus production in PTEC but not in MC. Cells were infected with SARS-CoV HSR1 at an MOI of 0.1. After 3 days, cultures were split and this procedure was repeated every 3 days up to four passages. Cultivation passages are indicated with a T, and the number in subscript indicates the serial passage. At each passage, 50% of the cells were seeded in fresh medium. The SARS-CoV copy number was calculated by real-time PCR of ORF-1b in cell supernatants collected prior to cell subculture. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log10/ml obtained in three independent experiments.
- FIG. 6.
Proportion of viral quasispecies in the M protein following serial subculturing of PTEC. The percentage is relative to 10 clones obtained from SARS-CoV HSR1-III passaged three times in Vero E6 cells and each time point from T0 to T4.
- FIG. 7.
Kinetics of SARS-CoV HSR1 replication in PTEC and Vero E6 cells infected with the supernatant obtained from the T0 culture prior to cell division and the T4 subculture. PTEC and Vero E6 cells were infected at MOIs of 0.03 and 0.006, respectively. Both PTEC and Vero E6 cells were also incubated with a 1:1 mixture of T0 and T4 supernatants containing 5 × 104 PFU/each. Replication kinetics were measured by real-time PCR of ORF-1b in the supernatant harvested every 24 to 48 h p.i. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log10/ml obtained in three independent experiments.
- FIG. 8.
Kinetics of viral replication of WT and mutant infectious clone (ic) in PTEC (A) and Vero E6 cells (B). Replication kinetics were measured by real-time PCR of ORF-1b in the supernatant harvested every 24 to 48 h p.i. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log10/ml obtained in two independent experiments. Infectious virus present in the PTEC supernatant 168 h p.i. (C) was determined in a Vero E6 plaque assay. Values represent the mean PFU ± standard deviation as log10/ml.
