Infectious Bronchitis Virus Infection Induces Apoptosis during Replication in Chicken Macrophage HD11 Cells

Figures

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  Figure 1. Susceptibility of HD11 to infectious bronchitis virus (IBV) Beaudette infection. (A) Cytopathic effects (CPE) observed upon IBV Beaudette infection by 24 and 36 h post-infection (h.p.i.), and mock-infected HD11 cells displayed no CPE. (B) Growth kinetics of IBV Beaudette in HD11 cells infected at 10 multiplicity of infection (MOI). Data are shown as the mean ± standard error of the mean (SEM) of three independent experiments. (C) Production of IBV Beaudette (fluorescein isothiocyanate, FITC) could be observed at 24 h.p.i., and mock-infected HD11 cells showed no fluorescence.

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  Figure 2. IBV Beaudette induces apoptosis in HD11 cells. (A) The role of IBV Beaudette in cell viability. HD11 cells were infected with IBV Beaudette at different MOIs and detected at the indicated times. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. The data are shown as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 versus control group (0 h). (B) Morphological changes. IBV Beaudette-infected cells were observed with condensed chromatin and nuclear fragmentation under fluorescence microscopy followed by Hoechst 33342 staining. (C) The apoptotic rate of cells. IBV Beaudette-infected cells (10 MOI) were subjected to flow cytometry at different times. Data are shown as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 versus control group (0 h).

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  Figure 3. Effects of IBV Beaudette infection on caspases in HD11 cells. (A) The activity of caspases in IBV Beaudette-infected cells. The caspases -3, -8 and -9 activity in HD11 cells infected with IBV at 10 MOI at the designed times were determined. The data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01 versus the control group (0 h). (B) Role of caspase inhibitors in cell viability. Cell viability was determined by CCK-8 assay: 20 μM of each caspase inhibitor was utilized to pre-treat cells for 2 h. Then, the treated and untreated cells were both infected with IBV Beaudette at an 10 MOI for 36 h. The data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01 versus IBV infection alone. (C) The effect of initiator caspase-8 or -9 on the activation of caspase-3: 20 μM of each caspase inhibitor was utilized to pretreat cells for 2 h. Then, the treated and untreated cells were infected with IBV at 10 MOI for 36 h. Caspase-3 activity was detected using a colorimetric assay kit. Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01 versus virus infection alone.

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  Figure 4. IBV Beaudette-induced apoptosis was regulated by the expression of Fas/Fas ligand (FasL) and the Bcl-2 family. (A,B) mRNA expression levels of Fas and FasL were detected in IBV Beaudette-infected cells for the indicated times. The data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01 versus control. (C,D) mRNA expression levels of Bcl-2 and Bax were detected in IBV Beaudette-infected cells for indicated times. The data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01 versus control. (E) The activity of caspase-9 was party blocked by caspase-8 inhibitor. Following incubation with Z-IETD-FMK for 2 h, the cells were infected with IBV Beaudette for 24 h. Caspase-9 activity was detected using a colorimetric assay kit. Data are shown as the mean ± SEM. * p < 0.05 versus virus infection alone.

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  Figure 5. Interplay between cell apoptosis and virus replication. (A) Following incubation with inhibitors for 2 h, the cells were infected with IBV Beaudette of 10 MOI for 36 h. After pre-treatment, virus titers were determined as log TCID50/mL. Data are shown as the mean ± SEM. * p < 0.05 versus virus infection alone. (B,C) A 30 μM concentration of NH4Cl was used to incubate HD11 cells for 2 h before infection, then cells were infected with IBV Beaudette of 10 MOI for 36 h, and the virus titer (B) and the rate of apoptotic cells (C) were separately tested. Data are shown as the mean ± SEM. ** p < 0.01 versus cells infected without NH4Cl. (D,E) UV germicidal light was utilized to inactivate IBV Beaudette for 30 min. UV-inactivated virus infected HD11 cells for 36 h; the virus titer (D) and the rate of apoptotic cells (E) were measured separately. Data are shown as the mean ± SEM. ** p < 0.01 versus UV-untreated IBV Beaudette.