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

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

Correlation of FMDV RNA load and IFN-γ, and –λ expression in micro-dissected tissue samples.

<p>A) Non-Vaccinated and B) Vaccinated. IFN-γ and -λ mRNA expression levels measured by qRT-PCR in micro-dissected nasopharyngeal mucosa. FMDV RNA load had a significant positive influence on IFN-γ (orange) and IFN-λ (purple) expression in vaccinated animals early after infection (24 hpi), as well as on IFN-λ in non-vaccinated animals that were viremic/ pre-clinical (Category II). Linear regression lines are shown with 95% confidence intervals (gray). Significant correlations are marked with asterisks (*).</p

Differential host-virus interactions in nasopharyngeal mucosa of non-vaccinated (A-C) versus vaccinated (D-F) cattle.

<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). Host cells expressing MHC II (purple) and/or CD11c (aqua) are present in the subepithelial compartment and interspersed within epithelium, but without co-localizing with viral protein. 10x magnification, scale bar 100μm. <b>B-C)</b> 40x magnification of region identified in (A), scale bars 25μm. <b>B)</b> Select channel views demonstrate lack of co-localization of FMDV VP1 (red) with CD11c (aqua)/ MHC II (purple)-double positive cells. <b>C)</b> Merge of all images shown in (B), scale bar 25 μm. <b>D)</b> FMDV VP1 (red) protein co-localizes with cytokeratin (green) in an epithelial erosion in dome region of follicle-associated epithelium of nasopharyngeal mucosa of vaccinated steer at 24 hpi (animal number 11). Regionally extensive infiltration by MHC II (purple) and/or CD11c (aqua)-expressing cells co-localizing and interspersing with viral antigen.10x magnification, scale bar 100μm. <b>E-F)</b> 40x magnification of region identified in (D), scale bar 25μm. <b>E)</b> Select channels demonstrating co-localization of FMDV VP1 (red) with CD11c (aqua), MHC II (purple). <b>F)</b> Merge of images shown in (E), scale bar 25 μm.</p

IFN mRNA expression in micro-dissected nasopharyngeal mucosa with systemic and local interferon bioactivity.

<p>A) Non-Vaccinated and B) Vaccinated. IFN-α, -β, -γ, and -λ mRNA expression levels were measured by qRT-PCR in micro-dissected tissue compartments of nasopharyngeal mucosa and reported as mean fold-induction. Non-vaccinated steers are grouped by disease progression: Category I = Pre-Viremic/Pre-Clinical, Category II = Viremic/Pre-Clinical, Category III Viremic/Clinical. Vaccinated steers are grouped by time of euthanasia as there was no viremia or clinical disease in these animals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143666#pone.0143666.t002" target="_blank">Table 2</a>). mRNA expression levels are defined as fold-changes normalized to housekeeping gene (GAPDH) and relative to a baseline established from similar analyses of tissues from non-infected cattle. IFN I/III bioactivity measured in nasopharyngeal whole-tissue macerates (IU/g) and serum (IU/ml) collected on the day of euthanasia for each animal category tabulated below X-axes. Epith = non-lymphoid epithelium, FAE = lymphoid follicle associated epithelium, LF = lymphoid follicle, SM = submucosa (see supplement 1 for micro-anatomic definitions).</p

Primary infection of nasopharyngeal mucosal epithelial cells is similar in non-vaccinated and vaccinated cattle.

<p>Microscopic distribution of FMDV structural (VP1) and non-structural (3D) protein in dorsal nasopharyngeal mucosa of non-vaccinated (A-C) and vaccinated (D-F) cattle at 24 hpi. Multichannel immunofluorescent technique. <b>A)</b> Nasopharyngeal mucosa of non-vaccinated steer at 24 hpi (animal number 3). FMDV VP1 (red) and 3D (aqua) proteins co-localize with cytokeratin (green) in foci of primary FMDV infection in superficial layer of follicle-associated epithelium. Scarce MHC II+ cells (purple) are present in the subepithelium. 20x magnification, scale bar 50μm. <b>B-C)</b> 40x magnification of region identified in (A). <b>B)</b> Select channel combinations demonstrating co-localization of FMDV VP1(red) and FMDV 3D (aqua) with cytokeratin (green), but not with MHC II (purple) <b>C)</b> Merge of all images shown in (B), scale bar 25μm. <b>D)</b> Nasopharyngeal mucosa of vaccinated steer at 24 hpi (animal number 11). FMDV VP1 (red) and 3D (aqua) proteins co-localize with cytokeratin (green) within follicle associated epithelium. Intra-epithelial MHC II+ cells (purple) are in close proximity to virus-infected epithelial cells. 20x magnification, scale bar 50μm. <b>E-F)</b> 40x magnification of region identified in (D). <b>E)</b> Select channel combinations demonstrating FMDV VP1 (red) and 3D (aqua) colocalization with cytokeratin (green)-expressing cells and with MHC II- expressing cells (purple). <b>E)</b> Merge of images shown in (F), scale bar 25μm.</p

Micro-anatomic distribution of FMDV RNA in nasopharyngeal mucosa of non-vaccinated and vaccinated animals determined by laser capture microdissection.

<p>* Sample weight calculated on basis of dissected sample volume and tissue density.</p><p>Micro-anatomic distribution of FMDV RNA in nasopharyngeal mucosa of non-vaccinated and vaccinated animals determined by laser capture microdissection.</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