Transcriptomic Analysis of Persistent Infection with Foot-and-Mouth Disease Virus in Cattle Suggests Impairment of Apoptosis and Cell-Mediated Immunity in the Nasopharynx

Abstract

In order to investigate the mechanisms of persistent foot-and-mouth disease virus (FMDV) infection in cattle, transcriptome alterations associated with the FMDV carrier state were characterized using a bovine whole-transcriptome microarray. Eighteen cattle (8 vaccinated with a recombinant FMDV A vaccine, 10 non-vaccinated) were challenged with FMDV A24 Cruzeiro, and the gene expression profiles of nasopharyngeal tissues collected between 21 and 35 days after challenge were compared between 11 persistently infected carriers and 7 non-carriers. Carriers and non-carriers were further compared to 2 naïve animals that had been neither vaccinated nor challenged. At a controlled false-discovery rate of 10% and a minimum difference in expression of 50%, 648 genes were differentially expressed between FMDV carriers and non-carriers, and most (467) had higher expression in carriers. Among these, genes associated with cellular proliferation and the immune response-such as chemokines, cytokines and genes regulating T and B cells-were significantly overrepresented. Differential gene expression was significantly correlated between non-vaccinated and vaccinated animals (biological correlation +0.97), indicating a similar transcriptome profile across these groups. Genes related to prostaglandin E2 production and the induction of regulatory T cells were overexpressed in carriers. In contrast, tissues from non-carrier animals expressed higher levels of complement regulators and pro-apoptotic genes that could promote virus clearance. Based on these findings, we propose a working hypothesis for FMDV persistence in nasopharyngeal tissues of cattle, in which the virus may be maintained by an impairment of apoptosis and the local suppression of cell-mediated antiviral immunity by inducible regulatory T cells.

Fig 1. Differential gene expression between non-carriers, carriers and naïve controls: Genes higher expressed in carriers.

The 100 probes with the largest difference in expression between non-carriers and carriers (out of a total of 867 with q<0.1) are shown ordered by decreasing difference. Genes that were expressed higher in carriers are shown in Fig 1, and genes that were higher expressed in non-carriers are shown in Fig 2. For each probe, the fold change relative to the naïve controls is shown on the x-axis with the vertical dashed line representing no change compared to the naïve animals. Fold changes in signal intensity between non-carriers and naïve controls are marked with blue squares, and the fold changes between carriers and naïve animals are marked with red circles. Filled blue or red symbols indicate a significant difference in intensity (q<0.1) compared to the naïve animals. The horizontal distance between each square and circle represents the difference in signal intensity between non-carriers and carriers, and the color of the label indicates the group in which the signal intensity was higher (blue for non-carriers, red for carriers). The difference between non-carriers and carriers is significant (q<0.1) for all probes shown, independent of whether the difference between each infected group and the controls is significant.

Fig 2. Differential gene expression between non-carriers, carriers and naïve controls: Genes higher expressed in non-carriers.

The 100 probes with the largest difference in expression between non-carriers and carriers (out of a total of 867 with q<0.1) are shown ordered by decreasing difference. Genes that were expressed higher in carriers are shown in Fig 1, and genes that were higher expressed in non-carriers are shown in Fig 2. For each probe, the fold change relative to the naïve controls is shown on the x-axis with the vertical dashed line representing no change compared to the naïve animals. Fold changes in signal intensity between non-carriers and naïve controls are marked with blue squares, and the fold changes between carriers and naïve animals are marked with red circles. Filled blue or red symbols indicate a significant difference in intensity (q<0.1) compared to the naïve animals. The horizontal distance between each square and circle represents the difference in signal intensity between non-carriers and carriers, and the color of the label indicates the group in which the signal intensity was higher (blue for non-carriers, red for carriers). The difference between non-carriers and carriers is significant (q<0.1) for all probes shown, independent of whether the difference between each infected group and the controls is significant.

Fig 3. Overrepresented Gene Ontology terms among genes that were higher expressed in carriers: Terms related to cellular proliferation.

Taken from the output of the GOrilla web tool. Fields are colored by p-value, from >10^-3 (white) to <10^-9 (red).

Fig 4. Overrepresented Gene Ontology terms among genes that were higher expressed in carriers: Terms related to the adaptive immune response.

Taken from the output of the GOrilla web tool. Fields are colored by p-value, from >10^-3 (white) to <10^-9 (red).

Fig 5. Heat map of signal intensities for selected probes.

The ordering of the probes (rows) and animals (columns) is based on an unsupervised cluster analysis. The associated dendrograms are shown to the left and above the heat map. The colors in the heat map represent centered and scaled intensity values. Cells with negative z-scores (intensities lower than the overall mean for any given probe) are shaded red and cells with positive z-scores (higher intensities) are shaded blue. The colored sidebar above the heat map indicates the persistence status of each animal (carriers are shown in red, non-carriers in blue and controls in black).