Chondrocytes Contribute to Alphaviral Disease Pathogenesis as a Source of Virus Replication and Soluble Factor Production

Figures

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  Figure 1. Murine chondrocyte cells are susceptible to Ross River virus (RRV) infection. Chondrocyte cells in the cartilage region of RRV-infected mice were stained positive for RRV envelope 2 (E2) RNA by fluorescence in situ hybridisation (green/FITC). To identify the chondrocyte cells, type II collagen immunostaining was performed to locate the cartilage region (red/Cy3). Tissue sections were also stained to better visualise cell morphology: DNA (blue/DAPI) and β-tubulin (magenta/Cy5). No FITC signal was observed in mock-infected mice. The experiment was also performed using a negative control probe to rule out any non-specific RNA probe binding. Images were captured at 20× and 60× magnifications.

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  Figure 2. Human chondrocyte cells are susceptible to Ross River virus (RRV) infection. (A) In vitro virus titers (PFU/mL) of RRV-infected human chondrocyte cells (blue) and human skeletal muscle (red) cells at time-points 0, 6, 12, 36, and 48 h post-infection (h.p.i.) with data represented as means of triplicate samples with SEM. (B) Characterisation of type II collagen expression in human chondrocyte cells (brown). Image taken at 10× magnification.

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  Figure 3. Upregulation of key pro-inflammatory genes and extracellular matrix breakdown genes during Ross River virus (RRV) infection in human chondrocyte cells (grey) and skeletal muscle cells (white) at 0, 24, 36, and 48 h post-infection (h.p.i.). Samples were normalised using values from mock-infected groups. (A) Relative gene expression of key pro-inflammatory markers. Statistical analysis was performed by two-way ANOVA statistical analysis with Bonferroni’s test. (B) Relative gene expression of articular cartilage components and associated breakdown markers. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 4. Soluble mediators present during Ross River virus (RRV) infection in human chondrocytes cells (grey) and skeletal muscle cells (white) primary cells at 0, 24, 36, and 48 h post-infection (h.p.i.). (A) Pro-inflammatory and (B) anti-inflammatory cytokine levels were measured using the Biorad Bio-Plex® multiplex system. Statistical analysis was performed by two-way ANOVA statistical analysis with Bonferroni’s test. * p < 0.05; ** p < 0.01, **** p < 0.0001.