CDV-Onderstepoort infection is independent of cell mem-

To investigate the role of cell membrane cholesterol for initiation of CDV-Onderstepoort infec-tion an experimental setup hat to be established that included a positive and a negative control.

Infectious bronchitis virus (IBV), an avian coronavirus, is related to Murine hepatitis virus, for which a dependence on cell membrane cholesterol for entry into susceptible cells has been demonstrated by Thorp and Gallagher (2004). In contrast toMurine hepatitis virus, IBV

pro-4.1. Role of cholesterol for initiation of CDV infection 71

Figure 4.2: Vero cell viability after MβCD treatment. Vero cells were treated with increasing MβCD concentrations for 30 min at 37^◦C, stained with PI and subsequently analysed by flow cytometry. The results are mean values of four independent, fourfold experiments with standard deviation.

ductively infects Vero cells, a culture system used for infection by CDV-Onderstepoort. A first experiment was designed to verify whether IBV infection, likeMurine hepatitis virusinfection, is dependent on cell membrane cholesterol. Furthermore, the minimum MβCD concentration which efficiently inhibits entry of viruses susceptible to cholesterol depletion had to be deter-mined. Therefore, Vero cells were treated with increasing MβCD concentrations and subse-quently infected by IBV. Infection by the avian coronavirus, as determined by flow cytometry, was already inhibited after pretreating the cells with 1 mM MβCD, and treatment with 7.5 mM MβCD resulted in approximately 75 % reduction of the number of infected cells (figure 4.3).

From the results of Fig. 4.1 and Fig. 4.3, we conclude that a reduction of the cholesterol content of about 45 % reduces the efficiency of infection by 75 %.

Infection by Vesicular stomatitis virus (VSV), or by VSV-G pseudotyped viruses, has been shown to be cholesterol independent (Thorp and Gallagher, 2004; Shah et al., 2006; Katzman and Longnecker, 2003). To show that a productive virus infection is not generally inhibited upon cholesterol depletion of the cell membrane, Vero cells treated with increasing MβCD concentrations were infected by VSV. Figure 4.3 shows that infection by VSV was not inhibited, even at MβCD concentrations as high as 15 mM. The number of infected cells was even slightly increased upon cholesterol depletion.

72 4. Results

Figure 4.3:Infection efficiencies of IBV, VSV and CDV-Onderstepoort on cholesterol depleted Vero cells.

Vero cells were treated with increasing MβCD concentrations for 30 min at 37^◦C and subsequently infected with IBV, VSV or CDV-Onderstepoort at an MOI of 0.1. VSV and IBV were prepared for flow cytometry 1 dpi, CDV-Onderstepoort 2 dpi. Infection rates at 0 mM MβCD were set to 100 %. Results are mean values of three independent experiments with standard deviation (standard deviations for some CDV values are not visible as they are smaller than the data point).

Similar to VSV, infection by CDV-Onderstepoort was found not to be inhibited by any MβCD concentration tested (figure 4.3). These results were confirmed when the infection efficiencies for IBV, VSV and CDV-Onderstepoort after cholesterol depletion were determined by fluores-cence microscopy (Fig. 4.4).

In the experiments described above, infection was performed at an MOI of 0.1, which was the highest possible MOI for CDV-Onderstepoort. To exclude that an inhibitory effect of the MβCD treatment was MOI dependent, Vero cells were treated with the highly effective MβCD concentration of 7.5 mM or left untreated and subsequently infected with CDV-Onderstepoort, or VSV and IBV as controls, at different MOIs. Figure 4.5 demonstrates that the effect of MβCD treatment was independent of the MOI used for all three viruses tested. IBV infection was always reduced upon depletion of cellular cholesterol prior to infection, while VSV as well as CDV-Onderstepoort infection were never inhibited. Again, results were confirmed by fluo-rescence microscopy (figure 4.6).These results demonstrate that CDV-Onderstepoort infection is not affected by a reduction of the cholesterol content in the cell membrane.

4.1. Role of cholesterol for initiation of CDV infection 73

Mock

0mM

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Figure 4.4:Infection efficiencies by CDV-Onderstepoort, IBV or VSV on cholesterol-depleted Vero cells.

Vero cells were treated with increasing MβCD concentrations for 30 min at 37^◦C and subsequently infected with CDV-Onderstepoort, IBV or VSV, respectively at an MOI of 0.1. IBV and VSV infected cells were prepared for immunofluorescence microscopy 1 dpi, CDV-Onderstepoort infected cells 2 dpi. Pictures were taken at 100 x magnification. Results are representative of three independent experiments.

74 4. Results

Figure 4.5: Infection of cholesterol-depleted Vero cells by CDV-Onderstepoort, IBV or VSV at different MOIs. Vero cells were treated with medium or with 7.5 mM MβCD for 30 min at 37^◦C and subsequently infected with CDV-Onderstepoort, IBV or VSV at different MOIs. Infection efficiencies for IBV and VSV were determined by flow cytometry 1 dpi, for CDV-Onderstepoort 2 dpi. Results are mean values of three independent experiments with standard deviation.

4.1. Role of cholesterol for initiation of CDV infection 75

Mock MOI 0.005 MOI 0.05

0mM

7.5mM

0mM

7.5mM

0mM

7.5mM

CDV

IBV

VSV

Figure 4.6: Infection of cholesterol-depleted Vero cells by CDV-Onderstepoort, IBV or VSV at different MOIs. Vero cells were treated with medium or 7.5 mM MβCD for 30 min at 37^◦C and subsequently infected by CDV-Onderstepoort, IBV or VSV at different MOIs. IBV and VSV infected cells were prepared for fluorescence microscopy 1 dpi, CDV-Onderstepoort infected cells 2 dpi. Pictures were taken at 100 x magnification. Results are representative of three independent experiments.

76 4. Results