Pathogenesis of virulent and attenuated foot-and-mouth disease virus in cattle

Figures

• 

  Fig. 1. Antemortem infection dynamics in steers following aerosol-inoculation with distinct strains of FMDV-A24-Cruzeiro: a FMDV-WT (n = 20) or b FMDV-Mut (n = 9). a & b FMDV RNA detection in serum, oral and nasal swabs was performed through qRT-PCR, and is presented as log10 genome copy numbers (GCN)/ul. Data presented are average values (mean +/− SEM) based on samples collected from all cattle included at each time point. Virus isolation (VI) was performed on BHK-21 cells and is presented as the percentage of VI positive samples per time point. High GCN/ul of RNA and VI percentage positivity immediately after inoculation indicates detection of residual inoculated virus. * X-axes scales are non-linear. For FMDV-Mut, nasal swab data is derived entirely from animals 699, 9106, 9107, 1003, 1024, and 1025

• 

  Fig. 2. Detection of FMDV-WT by immunomicroscopy in bovine nasopharynx at 6 h post aerosol-inoculation. a Low magnification view demonstrates tissue architecture of the caudal aspect of the dorsal soft palate including surface epithelium (Ep), epithelial crypts (Cr), lymphoid follicles (LF) of mucosa-associated lymphoid tissue, and secretory glands (Gl). Two regions of interest (ROI) are centered on an epithelial crypt with a prominent subepithelial lymphoid follicle: solid-lined box indicates ROI highlighted in 1b; dashed box indicates ROI highlighted in 1c. Immunohistochemistry (IHC) with anti-FMDV capsid monoclonal antibody, micropolymer alkaline phosphatase detection system with hematoxylin counterstain. b Earliest microscopic detection of FMDV consisting of few cells containing FMDV VP1 within a shallow depression within the wall of an epithelial crypt. IHC with anti-FMDV capsid monoclonal antibody, micropolymer alkaline phosphatase detection system. c Serial section of tissue shown in a-b demonstrating few cells containing FMDV VP1 (red) within epithelium (green) or adhered to epithelial surface of crypt. Multichannel immunofluorescence microcopy, (animal ID 1002, tissue ID Dorsal…

• 

  Fig. 3. Detection of FMDV-WT by immunomicroscopy in bovine nasopharynx at 12 h post aerosol-inoculation. a Cells containing FMDV VP1 antigen (red) are within the cytokeratin-positive epithelium (green). Epithelium overlies an expansive field of mucosa-associated lymphoid tissue with numerous cells containing CD11c (turquoise) and MHCII (purple). Dashed-lined box indicates region of interest (ROI) shown at higher magnification in 3b. b FMDV-containing cells express cytokeratin and are in the superficial-most layer of epithelium. These cells have morphology consistent with acantholytic degeneration including swelling and dissociation from adjacent cells. Multichannel immunofluorescence microcopy, (animal ID 1027, tissue ID Dorsal nasopharynx -Rostral) (magnification: a 10×, b 40×)

• 

  Fig. 4. At 24 hpa, distribution and morphology of primary infection is similar for FMDV-WT and FMDV-Mut. Microscopic distribution of FMDV structural protein VP1 (red) cytokeratin (green), and CD11c (turquoise) in nasopharyngeal mucosa of cattle infected with FMDV-WT (a-d) or FMDV-Mut (e-h). a & e Both viruses similarly infected superficial cytokeratin-positive nasopharyngeal mucosal epithelial cells. Dashed-lined boxes indicate regions of interest (ROI) shown at higher magnification in b-d, f-h. b-d and f-h Higher magnification and selective channel combinations demonstrate similar co-localization patterns for both viruses. FMDV VP1 (red) predominantly localizes within cytokeratin-positive epithelial cells (green), but not with CD11c (turquoise). Few infected cells contain neither cytokeratin nor CD11c (phenotype undetermined). Multichannel immunofluorescence microcopy, (FMDV-WT animal ID 930, tissue ID Dorsal nasopharynx –Rostral; FMDV-Mut animal ID 699, tissue ID Dorsal soft palate -Caudal) (magnification: a & e 20×, b-d & f-h 40×)

• 

  Fig. 5. Distribution and morphology of pulmonary infection was similar for FMDV-WT at 48hpa and FMDV-Mut at 72hpa. Microscopic distribution of FMDV structural protein VP1 (red), cytokeratin (green), and CD11c (turquoise) in lungs of cattle infected with FMDV-WT (a-d) or FMDV-Mut (e-h). a & e Between 48 and 72hpa both viruses generated poorly demarcated foci of infected cells, and similarly formed vesicle-like cavitations comprised of acantholytic cells and debris. Foci of infection were more abundant in animals infected with FMDV-WT. b-d and f-h Higher magnification and selective channel combinations demonstrate similar co-localization patterns for both viruses. FMDV VP1 (red) predominantly localizes within cytokeratin-positive pulmonary epithelial cells (green), but not with CD11c monocytoid cells (turquoise). Acantholytic cells are cytokeratin-positive. Few infected cells contain neither cytokeratin nor CD11c (phenotype undetermined). Multichannel immunofluorescence microcopy. (FMDV-WT animal ID 960, tissue ID Lung; FMDV-Mut animal ID 1027, tissue ID Lung) (magnification: a & e 10×, b-d & f-h 40×)

• 

  Fig. 6. FMDV generates microvesicles within the palatine tonsil of cattle during the clinical phase of disease. a Low magnification image demonstrates the architecture of a large tonsillar crypt delineated by cytokeratin-positive (green) squamous epithelium. Dashed box indicates region of interest (ROI) within crypt wall containing a focus of FMDV (red)-infected cells forming a microvesicle. ROI is featured at higher magnification in 6b-d including different detection channels in each panel. b Inclusion of FMDV VP1 (red) and cytokeratin (green) channels demonstrates disruption of epithelial architecture with cavitation/vesiculation. Many of the FMDV-containing cells are also cytokeratin-positive (epithelial cells). c Inclusion of FMDV VP1 (red), CD11c (turquoise), and MHC II (purple) demonstrates that some of the FMDV-containing cells are also individually- or double-positive for these markers of monocytoid, phagocytic, and antigen presenting cells. d Simultaneous viewing of all 4 channels demonstrates that within the vesicular cavity, FMDV-containing cells of distinct phenotypes are interspersed and in close proximity. Multichannel immunofluorescence microcopy. (animal ID 938, tissue ID…