Phenotyping and susceptibility of established porcine cells lines to African Swine Fever Virus infection and viral production

Figures

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  Figure 1. Analysis of membrane receptors in PAM, IPAM-WT, IPAM-CD163, WSL, CΔ2+ and COS-7 cells. Cells were incubated with different antibodies against CD14, CD163, CD169, SLAI, SLAII and SWC3 membrane receptors and the percentage of positive cells (a) or mean fluorescence intensity (MFI) (b) was analysed by FACS (n ≥ 4, performed in duplicate; mean ± S.D.). An IgG2b-FITC (IgG-FITC) and anti-mouse IgGs Alexa Fluor-488 (IgG-488) were used as negative controls for CD14 expression and the other receptors respectively.

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  Figure 2. Analysis of membrane and intracellular expression of CD163 on IPAM-CD163 cells. Cells were permeabilized (intracellular expression) or non-permeabilized (membrane expression) before and after sorting and incubated with mouse-anti-CD163 antibody following with an anti-mouse IgGs AlexaFluor-488. Cells were incubated with anti-mouse IgGs AlexaFluor-488 (IgG-488) as background of the cell line. The percentage of positive cells was analyzed by FACS (n = 2; mean ± S.D).

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  Figure 3. Analysis of viral protein expression in different porcine cell lines. Cells were infected with NHV/P68 (a) or Armenia/07 (b) isolate (MOI = 3) and at 18 hpi, infected cells were processed and prepared for western blot analysis. Membranes were incubated with antibodies against p32, p72 and p17 proteins; GAPDH was detected as a load control. Gels are representative of two independent experiments. M (mock); Inf (infected).

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  Figure 4. Analysis of PAM, IPAM-WT, CΔ2+ and WSL infected cells. Cells were infected with NHV/P68 (a) or Armenia/07 (b) ASFV isolates (MOI = 1) and at 18, 40 and 72 hpi, cells were processed for FACS analysis. The percentage of infected cells was detected with a specific Ab against the viral protein p72 (17LD3) following of an anti-mouse IgGs Alexa Fluor-488 (n ≥ 2, performed in duplicate; mean ± S.D.).

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  Figure 5. Analysis of viral protein expression in PAM and WSL at late times post-infection. Cells were infected with NHV/P68 (a) and Armenia/07 (b) isolates (MOI = 2) and viral protein expression was analyzed at 6, 12, 24, 48, 72 and 96 hpi with antibodies against p72, p32, p17 and p12 proteins. Gels are representative of two independent experiments.

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  Figure 6. Analysis of ASFV production in PAM and WSL. Cells were infected with NHV/P68 (a,b), Armenia/07 (c,d) and E70 (e,f) isolates (MOI = 0.2) and at indicated times post-infection, total virus (a,c,e) and extracellular virus (b,d,f) was recovered and titrated. The viral production is represented as plaque formation units (Pfu) (n ≥ 2; mean ± S.D.). y-axis is shown on a logarithmic scale.

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  Figure 7. Viral factory pattern in PAM. Cells were infected with NHV/P68 and Armenia/07 strains (MOI = 1), fixed at 16hpi and incubated with phalloidin-TRITC, anti-p72 antibody and DAPI to respectively stain actin filaments, viral particles and cellular and viral DNA. Z-slides images were taken by CLSM and represented as a maximum of z-projection. Arrowheads show the viral factories visualized by anti-p72 MAb and with DAPI to stain the viral DNA. Images are representative of two independent experiments.

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  Figure 8. Viral factory pattern in WSL. Cells were infected with NHV/P68 and Armenia/07 strains (MOI = 1), fixed at indicated times and incubated with phalloidin-TRITC, anti-p72 antibody and DAPI to stain respectively actin filaments, viral particles and cellular and viral DNA. Z-slides images were taken by CLSM and represented as a maximum of z-projection. Arrowheads show the viral factories visualized by anti-p72 MAb and with DAPI to stain the viral DNA. Images are representative of two independent experiments.