Canine Coronavirus Activates Aryl Hydrocarbon Receptor during In Vitro Infection
<p>Identifying CC<sub>50</sub> of CH223191 inhibitor at different doses and developing dose–response curve in A72 cells. (<b>a</b>) Dose–response curve of A72 cells treated with CH223191 at different concentrations (2, 5, 10 and 20 μM). After 24 h of treatment, cell viability was determined by TB staining while cells were attached to wells and counted under a light microscope. Significant differences between DMSO and CH223191-treated cells are indicated by probability <span class="html-italic">p</span>. * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01. (<b>b</b>) At 24 h after treatment, cells were stained with Giemsa and observed under a light microscope. (<b>c</b>) A72 cells treated with DMSO or with CH223191 (2 μM). Scale bar 100 µm.</p> "> Figure 2
<p>AhR inhibitor CH223191 increases cell viability during CCoV infection. (<b>a</b>) Dose–response curve of A72 cells treated with CH223191 at different concentrations (2, 5, 10 and 20 μM). After 24 h of treatment, cell viability was determined by TB staining while cells were attached to wells and counted under a light microscope. Significant differences between CCoV+DMSO and CCoV+CH223191-treated cells are indicated by probability <span class="html-italic">p</span>. * <span class="html-italic">p</span> < 0.05. (<b>b</b>) A72 cells infected with CCoV and treated or untreated with CH223191 at different concentrations (2, 5, 10 and 20 µM). At 24 h after treatment, cells were stained with Giemsa and observed under a light microscope. Scale bar 100 µm. (<b>c</b>) A72 cells infected with CCoV and treated or untreated with CH223191 (2 µM). At 24 h after treatment, cells were stained with Giemsa and observed under a light microscope. Scale bar 25 µm.</p> "> Figure 3
<p>AhR inhibitor CH223191 reduces morphological cell death signs during CCoV infection in A72 cells. Cells were infected with CCoV, in the presence or absence of CH223191. At 24 h p.i., cells stained by Giemsa were analyzed under a light microscope. Photomicrographs showing in unexposed infected groups, some cells with cell death features, such as an increase in intercellular spaces due to detachment from culture plate (arrow). In addition, morphological apoptotic marks, such as cell shrinkage (arrowhead), pyknosis and chromatin condensation (circle), were detected. In the presence of AhR inhibitor, all those cell death features were markedly diminished in CCoV-infected cells.</p> "> Figure 4
<p>A72 cells express AhR. (<b>a</b>) Canine fibrosarcoma cell line A72 expressed AhR. AhR inhibitor CH223191 noticeably decreased the expression of AhR. Scale bar 100 µm. (<b>b</b>) Bars represent the mean ratio generated from the integrated density (product of the area and mean intensity of fluorescence) of the AhR expression evaluated by ImageJ. Error bars represent standard deviation measurement. Significant differences between CCoV-infected cells and AhR-inhibitor-treated infected cells are indicated by probability <span class="html-italic">p</span>. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 5
<p>CCoV infection activates the expression of AhR in A72 cells. A72 cells were infected with CCoV at MOI of 0.05. At 24 h p.i., immunofluorescence staining for AhR was performed. (<b>a</b>) In CCoV-infected cells a significant increase in AhR expression was found. Scale bar 100 µm. (<b>b</b>) Bars represent the mean ratio generated from the integrated density (product of the area and mean intensity of fluorescence) of the AhR expression during CCoV infection evaluated by ImageJ. Error bars represent standard deviation measurement. Significant differences between CCoV-infected cells and AhR-inhibitor-treated infected cells are indicated by probability <span class="html-italic">p</span>. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 6
<p>AhR inhibitor inhibits both AhR and NP expression during CCoV infection in A72 cells. A72 cells were infected with CCoV at MOI of 5. At 24 h p.i., immunofluorescence staining for AhR and NP was performed. (<b>a</b>) In CCoV-infected cells a significant up-regulation of AhR and NP expression was detected. In some merged images of A72 cells, co-expression of NP and AhR expression was found (MERGE). Following infection, in the presence of AhR inhibitor CH223191, both AhR and NP expression was down-regulated. Scale bar 59 µm. (<b>b</b>) Bars represent the mean ratio generated from the integrated density (product of the area and mean intensity of fluorescence) of the NP expression during CCoV infection evaluated by ImageJ. Error bars represent standard deviation measurement. Significant differences between CCoV-infected cells and AhR-inhibitor-treated infected cells are indicated by probability p. ** <span class="html-italic">p</span> < 0.01. (<b>c</b>) Bars represent the mean ratio generated from the integrated density (product of the area and mean intensity of fluorescence) of the AhR expression during CCoV infection evaluated by ImageJ. Error bars represent standard deviation measurement. Significant differences between CCoV-infected cells and AhR-inhibitor-treated infected cells are indicated by probability <span class="html-italic">p</span>. *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 7
<p>AhR inhibitor decreases virus yield during CCoV infection in A72 cells. Cells were infected with CCoV in the presence or absence of AhR inhibitor CH223191. At 24 h p.i., virus yield was evaluated by RT-qPCR by the mean of a standard curve created plotting Log TCID<sub>50</sub>/mL against the C<sub>t</sub> number. Significant differences between CCoV-infected cells and AhR-inhibitor-treated infected cells are indicated by probability <span class="html-italic">p</span>. * <span class="html-italic">p</span> < 0.05.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures and Virus Infection
2.2. Cell Viability
2.3. Examination of Cell Morphology
2.4. Immunofluorescence Staining
2.5. Virus Infection
2.6. Viral Nucleic Acids Extraction Procedures
2.7. CCoV Viral Load Quantification by Real-Time Reverse Transcription PCR (RT-qPCR)
2.8. Statistical Analysis
3. Results
3.1. AhR Inhibitor Increases Cell Viability during CCoV Infection
3.2. AhR Inhibitor Reduces Morphological Cell Death Features during CCoV Infection in A72 Cells
3.3. A72 Cells Express AhR
3.4. CCoV Infection Activates the Expression of AhR in A72 Cells
3.5. AhR Inhibitor Inhibits Both AhR and NP Expression during CCoV Infection in A72 Cells
3.6. AhR Inhibitor Decreases Virus Yield during CCoV Infection in A72 Cells
3.6.1. CPE Evaluation
3.6.2. Standard Curve and Virus Quantification
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Host | Genus | CoVs | Model | Cell Type | References |
---|---|---|---|---|---|
Mouse | Betacoronavirus | M-CoV | In vitro | Bone-marrow-derived macrophage | [23] |
Mouse | Betacoronavirus | M-CoV | In vitro | Bone-marrow-derived dendritic cells | [23] |
Mouse | Betacoronavirus | M-CoV | In vitro | Liver (C57BL-6 mice) | [23] |
Mouse | Betacoronavirus | M-CoV | In vitro | Bone-marrow-derived macrophage | [24] |
Human | Alphacoronavirus | H-CoV-229E | In vitro | Human hepatoma (Huh7) | [22] |
Human | Alphacoronavirus | H-CoV-229E | In vitro | Human lung adenocarcinoma (A549) | [24] |
Human | Betacoronavirus | MERS-CoV | In vitro | Human lung adenocarcinoma (Calu-3) | [24] |
Human | Betacoronavirus | SARS CoV-1 | In vitro | Human hepatoma (Huh7) | [22] |
Human | Betacoronavirus | SARS-CoV-2 | In vitro | Primary human lung epithelium (NHBE) | [24] |
Human | Betacoronavirus | SARS-CoV-2 | In vitro | Human lung adenocarcinoma (A549) | [24] |
Human | Betacoronavirus | SARS-CoV-2 | In vitro | Human lung adenocarcinoma (Calu-3) | [24] |
Human | Betacoronavirus | SARS-CoV-2 | Patients | Nasal swabs | [24] |
Dog | Alphacoronavirus | CCoV-IIa | In vitro | Canine Fibrosarcoma (A72) | This study |
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Cerracchio, C.; Serra, F.; Amoroso, M.G.; Fiorito, F. Canine Coronavirus Activates Aryl Hydrocarbon Receptor during In Vitro Infection. Viruses 2022, 14, 2437. https://doi.org/10.3390/v14112437
Cerracchio C, Serra F, Amoroso MG, Fiorito F. Canine Coronavirus Activates Aryl Hydrocarbon Receptor during In Vitro Infection. Viruses. 2022; 14(11):2437. https://doi.org/10.3390/v14112437
Chicago/Turabian StyleCerracchio, Claudia, Francesco Serra, Maria Grazia Amoroso, and Filomena Fiorito. 2022. "Canine Coronavirus Activates Aryl Hydrocarbon Receptor during In Vitro Infection" Viruses 14, no. 11: 2437. https://doi.org/10.3390/v14112437