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

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

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

Pathogenesis of Primary Foot-and-Mouth Disease Virus Infection in the Nasopharynx of Vaccinated and Non-Vaccinated Cattle

<div><p>A time-course pathogenesis study was performed to compare and contrast primary foot-and-mouth disease virus (FMDV) infection following simulated-natural (intra-nasopharyngeal) virus exposure of cattle that were non-vaccinated or vaccinated using a recombinant adenovirus-vectored FMDV vaccine. FMDV genome and infectious virus were detected during the initial phase of infection in both categories of animals with consistent predilection for the nasopharyngeal mucosa. A rapid progression of infection with viremia and widespread dissemination of virus occurred in non-vaccinated animals whilst vaccinated cattle were protected from viremia and clinical FMD. Analysis of micro-anatomic distribution of virus during early infection by lasercapture microdissection localized FMDV RNA to follicle-associated epithelium of the nasopharyngeal mucosa in both groups of animals, with concurrent detection of viral genome in nasopharyngeal MALT follicles in vaccinated cattle only. FMDV structural and non-structural proteins were detected in epithelial cells of the nasopharyngeal mucosa by immunomicroscopy 24 hours after inoculation in both non-vaccinated and vaccinated steers. Co-localization of CD11c⁺/MHC II⁺ cells with viral protein occurred early at primary infection sites in vaccinated steers while similar host-virus interactions were observed at later time points in non-vaccinated steers. Additionally, numerous CD8⁺/CD3^- host cells, representing presumptive natural killer cells, were observed in association with foci of primary FMDV infection in the nasopharyngeal mucosa of vaccinated steers but were absent in non-vaccinated steers. Immunomicroscopic evidence of an activated antiviral response at primary infection sites of vaccinated cattle was corroborated by a relative induction of interferon -α, -β, -γ and -λ mRNA in micro-dissected samples of nasopharyngeal mucosa. Although vaccination protected cattle from viremia and clinical FMD, there was subclinical infection of epithelial cells of the nasopharyngeal mucosa that could enable shedding and long-term persistence of infectious virus. Additionally, these data indicate different mechanisms within the immediate host response to infection between non-vaccinated and vaccinated cattle.</p></div

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

Tissue distribution of FMDV in vaccinated steers.

<p>Numbers represent log₁₀ FMDV genome copy numbers (GCN)/mg of tissue or GCN/μl serum. <b>Bold</b> 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: 1.53 log₁₀ FMDV GCN/mg of tissue, <0.1 log₁₀ GCN/ μl serum (corresponding to an assay detection limit of 1.57 log₁₀ FMDV GCN/ml serum)</p><p>Tissue distribution of FMDV in vaccinated steers.</p