Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis

Figures

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  Figure 1. Experimental timeline for monitoring FMDV infection of porcine DSP. (A) Experimental timeline for monitoring FMDV infection of porcine DSP cultured in monolayers. Porcine DSP cells were propagated in monolayers and were infected with FMDV O Cl2.2 at MOI 0.01, 0.1, or 1, or a placebo-conditioned medium. Culture supernatants were collected every 2 or 3 days during 112 dpi as illustrated above. (B) Experimental timeline for monitoring FMDV infection of porcine DSP cultured in multilayers at the air-liquid interface. Porcine DSP cells were propagated in multilayers on porous membrane inserts at the air-liquid interphase and were infected with FMDV O Cl2.2 at MOI 1, or a placebo-conditioned medium. Insert upper supernatants were collected every 2 or 3 days during 39 dpi as illustrated above. Analyses were performed on collected supernatants: virus isolation (squares) and RT-qPCR (circles) immunofluorescence (triangles). Immunochemistry analyses were performed on fixed inserts (hexagons) and CPE observations were also performed (stars), at indicated days postinfection.

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  Figure 2. Oropharyngeal tonsils and dorsal soft palate. DSP and oropharyngeal tonsils were collected from 4 to 6-month-old American Yorkshire swine. Histological analysis, HES staining.

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  Figure 3. Porcine DSP cells characterization. (A) Porcine DSP morphological observation. DSP cells were cultured in monolayers and were observed under brightfield microscopy (x100). (B) Cytokeratin staining. DSP was cultured in monolayers on High ibiTreat Ibidi chamber slides. Cell nuclei were stained with Hoechst (blue) and cells were visualized by fluorescence microscopy using a DMi8 microscope. Staining with specific mouse cytokeratin antibody and a goat anti-mouse Alexa 488 (green). DSP staining was compared to the ZZ-R127 epithelial cell line, known to strongly express cytokeratin, and the BUcEC endothelial cell line, known to not express cytokeratin. (C) Occludin staining. DSP was stained with a specific rabbit occludin antibody and a goat anti-rabbit Alexa 488 (green). DSP staining was compared to the BUcEC endothelial cell line, which is known to not express occludin. (D) Vimentin staining. DSP was stained with a specific mouse vimentin antibody and a goat anti-mouse Alexa 488 (green). DSP staining was compared to the ZZ-R127 epithelial cell line, which is known to strongly express cytokeratin. (E) αVβ6 integrin staining. DSP was stained with a specific mouse cytokeratin antibody…

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  Figure 4. Assessment of porcine DSP susceptibility to a panel of viruses involved in the differential diagnosis of FMDV. (A) Porcine DSP susceptibility to FMDV strains. Porcine DSP, bovine DSP, ZZ-R127, and IBRS-2 were cultured in 48-well plates and infected with FMDV Asia1/PAK/2011, SAT1/NIG/2015, SAT3/ZIM/1981, O/OMN/2020, O/FRA/2001, O/MUR/2016, O/ALG/2018, or A/TUN/2017 strain. At 48 h postinfection, CPE was visually determined and supernatants were collected in order to perform viral titration in BHK-21 cells. Virus titers are expressed as TCID50/mL. (B) Porcine DSP susceptibility VSV, SVV, and SVDV strains. Porcine DSP, bovine DSP, ZZ-R127, and IBRS-2 were cultured in 48-well plate and infected with VSV/IND/1942, VSV/NJ/1964, SVV/CA/2001, SVV MN/1988, SVDV/FRA/1973, or SVDV/ITL/2008 strain. At 48 h postinfection, CPE was visually determined and supernatants were collected in order to perform viral titration in porcine DSP cells. Virus titers are expressed as TCID50/mL.

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  Figure 5. Assessment of porcine DSP ability to produce type-I IFN. Porcine DSP and bovine DSP were cultured in 6-well plate and incubated with FMDV O Cl2.2 or conditioned medium. At 6, 12, and 24 hpi, cellular RNA was extracted in order to perform rtRT-PCR targeting type-I IFN response components. (A) IFNβ; (B) IFNα; (C) MX-1; and (D) PKR.

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  Figure 6. Monitoring of FMDV infection of porcine DSP cultured in monolayers. Porcine DSP was cultured in monolayers and was infected with FMDV O Cl2.2 at MOI 0.01, 0.1, or 1, or a placebo-conditioned medium. (A) CPE observation at 48 hpi. (B) CPE evolution during the first 7 dpi. CPE was visually estimated using brightfield microscopy. (C) Infectious FMDV detection by viral isolation. Viral isolation on IBRS-2 sensitive cells was performed using collected supernatants to detect infectious FMDV. CPE on IBRS-2 after 48 h of incubation with supernatants was visually estimated. The results presented here concern culture supernatants collected during the first 23 days postinfection. (D) FMDV 3Dpol antigen detection by immunofluorescence. IBSR-2 96-well plates used for viral isolation were fixed with 4% paraformaldehyde and then permeabilized with Triton X-100 before being stained with a specific mouse 3Dpol antibody and a goat anti-mouse Alexa 488 (green). Cell nuclei were stained with Hoechst (blue) and cells were visualized by fluorescence microscopy using a DMi8 microscope. Results were arbitrarily classified according to an index ranging from 0 to 3. 0 indicates no fluorescence, 1…

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  Figure 7. DSP cells multilayer characterization. Porcine DSP was cultured in multilayers at the air-liquid interface for 6 weeks. Some inserts were sacrificed to evaluate the multilayer depth. To this purpose, inserts were fixed in formalin and ethanol and then stained with HES. Stained inserts were observed by microscopy to estimate the number of cell layers.

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  Figure 8. Monitoring of FMDV infection of porcine DSP cultured in multilayers at the air-liquid interface. DSP was cultured in multilayers at the air-liquid interface for 6 weeks before being infected with FMDV O Cl2.2 at MOI 1, or a placebo-conditioned medium. CPE was visually estimated almost daily during the first 2 weeks postinfection. (A) CPE evolution on DSP multilayers. Microscopic CPE observation was pictured just before infection, at 24 hpi, 48 hpi, 7 dpi, and 14 dpi for infected and MOCK conditions. (B) Infectious FMDV detection by viral isolation. Viral isolation on IBRS-2 sensitive cells was performed using collected supernatants to detect infectious FMDV. CPE on IBRS-2 was visually estimated after 48 h of incubation with supernatants passaged once in IBRS-2. The results presented here concern culture supernatants collected during the first 11 days postinfection. (C) FMDV 3Dpol antigen detection by immunofluorescence. IBSR-2 96-well plates used for viral isolation were fixed with 4% paraformaldehyde and then permeabilized with Triton X-100 before being stained with a specific mouse 3Dpol antibody and a goat anti-mouse Alexa 488. Cell nuclei were stained with Hoechst and cells…