Model of persistent foot‐and‐mouth disease virus infection in multilayered cells derived from bovine dorsal soft palate

Figures

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  Figure 1. Schematic draw of a permeable insert used to propagate multilayers of bovine dorsal soft palate cells. Insert (a); upper compartment (b); multilayer of bovine dorsal soft palate cells (c); cell culture medium (d); porous membrane (e); lower compartment (f) and well (g) of a 12‐well plate

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  Figure 2. Experiment timeline and FMDV detection. Bovine dorsal soft palate cells were propagated in multilayers on porous membrane inserts in the air‐liquid interphase and were infected with FMDV O Cl2.2, or placebo. In experiment 1 (Exp 1, empty legends) and 2 (Exp 2, light grey legends), cells derived from male and female cattle, respectively, and were infected at an MOI of 0.01. In experiment 3 (Exp 3, dark grey legends) cells derived from the same female cattle and were infected at an MOI of 1. Cell culture medium and cells were analysed for the presence of FMDV, by virus isolation (triangles), RT‐qPCR (circles) or immunohistochemistry (squares), at indicated days post‐infection

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  Figure 3. Bovine dorsal soft palate cells express cytokeratin, vimentin and integrin αVβ6 after passage in vitro. Bovine dorsal soft palate cells were isolated by protease digestion, filtration and removal of rapidly adherent cells. After freezing, thawing and three to five passages in flasks, cells were propagated in monolayers on Lab‐Tek chamber slides and stained with monoclonal antibodies specific for cytokeratin (green a), vimentin (green, b) or integrin αVβ6 (red, c) and secondary antibodies conjugated with fluorescein isothiocyanate or Alexa Flour® 647. Cell nuclei were stained with DAPI (blue, a and b) and cells were visualized by confocal laser scan microscopy (in a ZEISS LSM700 microscope) [Colour figure can be viewed at http://www.wileyonlinelibrary.com/]

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  Figure 4. Bovine dorsal soft palate cells form multilayers at the air‐liquid interface in vitro. Bovine dorsal soft palate cells were propagated on porous membrane inserts. a) Cross‐section of the multilayers (double arrow) lying on PTFE membrane as visualized by light microscopy; HES stain; bar = 20 µm. b) Cross‐section of the upper layer as visualized by transmission electron microscopy: tight junction (arrow) between two cells; (N: nucleus) at the upper layer; one of the cells displays apical microvilli (*); bar = 500 nm. c) Cross‐section of the multilayers after antibody staining against cytokeratin (green) and visualization by a Nikon eclipse Ts2R microscope (bar = 50 µm) [Colour figure can be viewed at http://www.wileyonlinelibrary.com/]

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  Figure 5. RNA transcripts related to tight junctions, FMDV receptor integrins and cytokeratins. Bovine dorsal soft palate cells were isolated and propagated in multilayers on porous membrane inserts in the air‐liquid interface. After five weeks of culture, membranes were infected with FMDV at MOI 0.01 or MOI 1 or with uninfected cell lysate as placebo. Cells were harvested at indicated time points, before (0 hr, 0h) and post‐infection (24 hr hpi, 28 days dpi) and total RNA was extracted. Messenger RNA was thereafter detected by RNA sequencing on the Ion S5XL system using the Ion 530 OT2 and Chip kit (Life Technologies). Gene transcripts associated with a) tight junctions (CLN, OCLN, TCF3, TCF7L1), b) FMDV‐associated integrin chains (ITGAV, ITGB1, ITGB3, ITGB8) and c) cytokeratin (KRT) expression are expressed as variance stabilizing transformed (vs.t) read counts. Each data point represents a technical replicate that derived from duplicate analyses of two biological replicates

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  Figure 6. Protein expression related to FMDV receptor‐associated integrins. Bovine dorsal soft palate cells were isolated and propagated in multilayers on porous membrane inserts in the air‐liquid interface. After five weeks of culture, membranes were infected with FMDV at MOI 0.01 or with uninfected cell lysate as placebo. Cells were harvested at indicated time points, before and after infection. Proteins were thereafter extracted, separated by SDS‐PAGE and analysed by LC‐MS/MS using an EASY‐nLC II coupled to a LTQ Orbitrap‐Velos mass spectrometer (Thermo Fisher Scientific). ITGB6 and ITGB8 were not detected

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  Figure 7. FMDV infection of multilayer cultures of bovine dorsal soft palate cells induces transient CPE in vitro. Bovine dorsal soft palate cells were isolated and propagated in multilayers on porous membrane inserts in the air‐liquid interphase. After five weeks of culture, membranes were infected with FMDV at MOI 0.01 or MOI 1 or with uninfected cell lysate as placebo (Mock). A limited cytopathic effect was observed by light microscopy, at 24 hpi and up to 7 dpi, respectively, but not in mock‐infected inserts. Bar = 100 µm

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  Figure 8. Multilayers of bovine dorsal soft palate cells show limited lysis after FMDV infection and carry persistent FMDV antigen. After five weeks of culture, dorsal soft palate multilayers were infected with FMDV at MOI 0.01 or MOI 1 or with uninfected cell lysate as placebo. At 24 hpi (a‐f), infection resulted in multifocal cell detachment and loss, with foci of sparing of several layers (a, infection at MOI 0.01, Exp 2, HES stain) and foci of loss of the deeper layers (b, infection at MOI 1, Exp 3, HES stain), in contrast to the mock‐infected membranes that did not show lysis (c, HES stain). The cell morphology in infected and control multilayers was similar (a‐c). FMDV antigen was detected by immunohistochemistry (brown labelling) at the upper, middle and lower levels of the multilayers infected at MOI 0.01 (d) and MOI 1 (e), but not in the control multilayers (f). At 28 dpi (g‐l), the infected and control multilayers were 1 to 4 cells thick with similar morphology (g, MOI 0.01, Exp 2; h, MOI 1, Exp 3; i, control; HES stain). FMDV antigen was detected by IHC (brown labelling) in a few cells at all levels of the multilayers infected at MOI 0.01 (j) and MOI 1 (k), but not in the…

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  Figure 9. Expression of FMDV non‐structural protein 3D in multilayers of bovine dorsal soft palate after FMDV infection. Expression of viral non‐structural protein 3D was detected by immunohistochemistry in the cytoplasm of cells after infection at an MOI of 1 at 24 hpi (a) and at 28 dpi (b) but not in the control multilayers (c). A vesicle‐like structure with intercellular oedema (star) and an image consistent with suprabasilar clefting (arrowhead) was detected 24h after infection at an MOI of 1; the basal cells and the other cells at the edges of the cleft expressed viral 3D. Bar = 20 µm [Colour figure can be viewed at http://www.wileyonlinelibrary.com/]

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  Figure 10. FMDV RNA and live FMDV can be recovered from persistently infected bovine dorsal soft palate cells that are propagated in vitro. Bovine dorsal soft palate cells were isolated and propagated in multilayers on porous membrane inserts in the air‐liquid interface. After five weeks of culture, membranes were infected with FMDV at MOI 0.01 (a) or MOI 1 (b) or with uninfected cell lysate as placebo. The cells were repeatedly washed with cell culture medium. The wash from 1–6 inserts, collected at indicated time points, was analysed for presence of the 3D polymerase coding region of FMDV RNA by RT‐qPCR or was serially diluted from 1:10 and inoculated on susceptible IBRS‐2 cultures. After three days of incubation, cells were fixed, stained with methylene blue and the TCID50 calculated based on CPE

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  Figure 11. The proportion of bovine dorsal soft palate cells that carry persistent FMDV antigen is low. Multilayers of dorsal soft palate cells were infected with FMDV at an MOI of 0.01 (Exp 1 and 2) or at an MOI of 1 (Exp 3). The proportion of FMDV‐positive cells was analysed by immunohistochemistry and cell counting, on two sections per insert. At 24 hpi, 36% of the cells were FMDV‐positive after infection at an MOI of 1, while 1 to 3% of the cells were FMDV‐positive at an MOI of 0,01. At 28 dpi and regardless of MOI, less than 3% off cells were infected