Regionally extensive micro-vesiculation within epithelial crypts on the tonsil of the soft palate at 78 hpi detected by multichannel immunofluorescence.

<p><b>A)</b> Intra-epithelial microvesicles containing large quantities of FMDV structural (VP1; red) and non-structural (3D; turquoise) protein co-localized with cytokeratin (green) positive epithelial cells. 10× magnification, scale bar 100 µm. <b>B)</b> Serial section of region identified in A. FMDV VP1 protein (red) detected within cytokeratin (green) positive epithelial cells encircling an intra-epithelial microvesicle with acantholytic FMDV VP1/cytokeratin double positive cells detected within the vesicle lumina. CD172a (turquoise) and CD8 (purple; presumptive NK cells) leukocytes detected within and around the epithelial lesion, but without co-localization. 20× magnification, scale bar 50 µm. <b>C)</b> Cytokeratin-18 (turquoise) expressing M-cells in close proximity of FMDV VP1 (red) infected cytokeratin (green) positive epithelial cells within the tonsil of the soft palate. 40× magnification, scale bar 100 µm.</p

Tissue distribution of FMDV during viremic phase of infection following IOP inoculation.

<p>Numbers represent Log₁₀ genome copy numbers (GCN)/mg of tissue. FMDV RNA content in serum is expressed as Log₁₀ FMDV RNA copies/µl to facilitate comparison with viral content in tissues. Bold numbers indicate that samples were positive for both FMDV RNA (qRT-PCR) and virus isolation, (+) indicates that virus isolation was positive but FMDV RNA content was below the limit of detection, (-) indicates double negative samples. Limits of detection: tissue samples  = 2.52 Log₁₀ GCN/mg, serum≤0.1 Log₁₀ GCN/µl (corresponding to an assay detection limit of 2.68 Log₁₀ GCN/ml of serum).</p><p>Tissue distribution of FMDV during viremic phase of infection following IOP inoculation.</p

Multichannel immunofluorescent detection of FMDV structural (VP1) and non-structural (3D) protein in porcine paraepiglottic tonsils at 24 hpi.

<p>A) FMDV VP1 (red) and 3D (turquoise) proteins co-localize with cytokeratin (green) in regionally expanding foci of primary FMDV infection within reticular crypt epithelium of the paraepiglottic tonsil. 10× magnification, scale bar 100 µm. B) Serial section of region identified in (A). Localization of FMDV VP1(red) is restricted to cytokeratin-positive epithelial cells (green); leukocytes expressing CD172a (turquoise) and CD8 (purple; presumptive NK cells) are interspersed amongst virus-infected cells. 40× magnification, scale bar 50 µm. C) Serial section of region identified in (A–B). Cytokeratin-18 expressing M-cells (turquoise) localized within segments of epithelium containing FMDV VP1-positive cells (red), but without co-localization. 40× magnification, scale bar 50 µm.</p

Multichannel immunofluorescent detection of FMDV structural (VP1) protein in the tonsil of the soft palate at 48 hpi.

<p><b>A)</b> Cluster of FMDV VP1(red) positive epithelial cells (green) in a developing microvesicle within reticular crypt epithelium of the tonsil of the soft palate. Leukocytes expressing CD172a (turquoise) and CD8 (purple; presumptive NK cells) are interspersed within crypt epithelium and are present in larger numbers in adjacent (sub-epithelial) tissue. 10× magnification, scale bar 100 µm. <b>B)</b> Serial section of region identified in (A). Intraepithelial microvesicle spanning basal and spinous layers of crypt epithelium. FMDV VP1(red)/cytokeratin (green) double-positive cells are present within the vesicle and surrounding epithelium with MHC II(turquoise)-expressing cells in close proximity. 40× magnification, scale bar 25 µm. <b>C)</b> Serial section of region identified in (A–B). Cytokeratin-18 (turquoise) expressing M-cells are localized within crypt epithelium in close proximity of FMDV VP1(red) positive epithelial cells (green), but without co-localization. 40× magnification, scale bar 25 µm.</p

Tissue distribution of FMDV during viremic phase of infection following contact exposure.

<p>Numbers represent Log₁₀ genome copy numbers (GCN)/mg of tissue. FMDV RNA content in serum is expressed as Log₁₀ FMDV RNA copies/µl to facilitate comparison with viral content in tissues. Bold numbers indicate that samples were positive for both FMDV RNA (qRT-PCR) and virus isolation, (+) indicates that virus isolation was positive but FMDV RNA content was below the limit of detection, (-) indicates double negative samples. Limits of detection: tissue samples  = 2.52 Log₁₀ GCN/mg, serum≤0.1 Log₁₀ GCN/µl (corresponding to an assay detection limit of 2.68 Log₁₀ GCN/ml of serum).</p><p>Tissue distribution of FMDV during viremic phase of infection following contact exposure.</p

Tissue distribution of FMDV during the pre-viremic phase of infection following IOP inoculation.

<p>Numbers represent Log₁₀ genome copy numbers (GCN)/mg of tissue. Bold numbers indicate that samples were positive for both FMDV RNA (qRT-PCR) and virus isolation, (+) indicates that virus isolation was positive but FMDV RNA content was below the limit of detection, (-) indicates double negative samples. Limit of detection: 2.52 Log₁₀ GCN/mg of tissue.</p><p>* Visceral organs included liver, spleen, kidney, pancreas, ileum and Peyer's patches. Other tissues analyzed with consistently negative results were epiglottis, larynx, pharyngeal-diverticulum, salivary glands, adrenal glands, bone marrow.</p><p>Tissue distribution of FMDV during the pre-viremic phase of infection following IOP inoculation.</p

Multichannel immunofluorescent detection of FMDV capsid protein (VP1) in porcine paraepiglottic tonsils at 6 hpi (A) and 12 hpi (B–C).

<p><b>A)</b> At 6 hpi FMDV VP1 is localized to individual cells within reticular crypt epithelium of the paraepiglottic tonsil. Virus antigen (red) is detected within few cytokeratin-positive epithelial cells (green) and CD172a-expressing non-lymphoid leukocytes (turquoise). 40× magnification, scale-bar 25 µm. <b>B and C)</b> At 12 hpi, foci of multiple FMDV VP1-positive cells are detected within similar regions of reticular crypt epithelium of the paraepiglottic tonsil. Detection of virus antigen (red) is restricted to cytokeratin-positive epithelial cells (green) in close proximity of CD172a-expressing leukocytes (turquoise). B: 20× magnification, scale bar 50 µm. C: 40× magnification, scale bar 25 µm.</p

Additional file 1: Table S3. of Proof-of-concept study: profile of circulating microRNAs in Bovine serum harvested during acute and persistent FMDV infection

Cattle serum samples used for the miRNA profiling study. (DOCX 12 kb

Nasopharyngeal primary infection sites of vaccinated cattle are infiltrated by CD8⁺/CD3^- (presumptive NK) cells at 24 hpi.

<p>Multichannel immunofluorescent technique. <b>A)</b>. FMDV VP1 (red) protein within cytokeratin⁺ cells (green) in epithelial crypt of the nasopharyngeal mucosa of non-vaccinated steer at 24 hpi (animal number 3). CD8⁺ (aqua)/ CD3⁺ (purple) double-positive CTLs are present in the subepithelial compartment amongst larger populations of CD8^-/CD3⁺ T-lymphocytes. 20x magnification, scale bar 50μm. <b>B)</b> 40x magnification of region identified in (A), demonstrating consistent co-localization of CD8 (aqua) with CD3 (purple). Scale bars 25μm. <b>B)</b> Select channels of image shown in (B). CD3⁺ cells (purple) include single-positive (T-lymphocytes) or CD8⁺/CD3⁺ double-positive (CTLs). CD8 (aqua) is exclusively detected in combination with CD3 (purple). Scale bars 25 μm. <b>D)</b> FMDV VP1 (red) protein co-localize with cytokeratin (green) in a focal surface erosion within follicle-associated epithelium of nasopharyngeal mucosa of vaccinated steer at 24 hpi (animal number 11). A distinct population of cells defined as presumptive NK-cells based on a CD8⁺ (aqua)/CD3^- (purple) phenotype is present in submucosal and epithelial compartments surrounding the focus of infection. A smaller population of CTLs (CD8⁺/CD3⁺) is present amongst abundant non-CTL T-lymphocytes (CD8^-/CD3⁺). 20x magnification, scale bar 50μm. <b>E)</b> Higher magnification of region identified in showing CD8⁺/CD3^- cells (aqua) representing presumptive NK cells in close proximity of FMDV infected focus. 40x magnification, scale bars 25 μm <b>F)</b> Individual channels of image shown in (E) demonstrating inconsistent co-localization of CD8 (aqua) and CD3 (purple). Scale bar 25 μm.</p

Antemortem infection dynamics in non-vaccinated and vaccinated steers following intra-nasopharyngeal inoculation with FMDV A24 Cruzeiro.

<p>A) Non-vaccinated steers, n = 10. B) Vaccinated steers, n = 6. FMDV RNA detection in serum, oral and nasal swabs was performed through qRT-PCR and is presented as log₁₀ genome copy numbers (GCN)/ml. Data presented are average values (mean +/- SD) based on samples collected from all cattle included at each time point. “p.i.” (“post-inoculation”) on time scale indicates swab samples harvested directly following inoculation.</p