Single-Cell Sequencing Yields Insights in the Evolution of Foot-and-Mouth Disease Virus Persistent Infection

Figures

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  Figure 1. Comprehensive FMDV-persistent infection map by single-cell sequencing. (A) Violin diagram of the quality control of single-cell sequencing. Dot diagram (B) and the heat map (C) of the gene clustering of cell subpopulations. tSNE displayed subclusters after cluster analysis (D). Heat map of the genes in the first nine principal components (E). UMAP displayed subclusters after cluster analysis (F).

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  Figure 2. Reconstruction of persistent infecting trajectory through pseudotime analysis. The genes used for the proposed pseudotime trajectory analysis after screening were identified (A). DDRTree method was used to show the distribution of pseudotime trajectory in two dimensions (B). The developmental trajectory of the cells is distinguished by the shade of color.

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  Figure 3. Characterization of the functional enrichment response during FMDV-persistent infection. Bar (A) and dot (B) diagram of the GO enrichment of BHK-Op. The dot from small to big corresponds to a small to large amounts of gene expression, and the color from blue to red corresponds to a low to high abundance of gene expression. Chord diagram (C) showed the relationship between genes and individual GO items. Bar (D) and dot (E) diagram of the KEGG enrichment of BHK-Op. The dot from small to big corresponds to a small to large amounts of gene expression, and the color from blue to red corresponds to a low to high abundance of gene expression. Scatterplot of gene dispersion (F) labeled the top fifteen genes with the greatest amount of variation. In two dimensions, the expression levels of FOS (G), JUN (H), and four other randomly selected genes (I) were displayed. The color light to dark corresponds to a low to high abundance of gene expression.

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  Figure 4. MAPK signaling pathway was activated under the infection of FMDV with reliance on viral replication. The protein expression of the marker proteins in MAPK signaling pathway after the infection of FMDV (A) or UV-treated FMDV (B) was confirmed by Western blot. The effects of MAPK/ERK signaling pathway inhibitor U0126-EtOH-EtOH on RNA levels (C), protein levels (D), and infectious FMDV virus titers (E) and the effects of MAPK/p38 signaling pathway inhibitor SB202190 on RNA levels (F), protein levels (G), and infectious FMDV virus titers (H). The intensity of pERK, p38, and p-JNK relative to GAPDH was measured by ImageLab software. GAPDH was served as the internal reference to normalize data. Three independent replicates were conducted for each sample. Data are expressed as the means ± SE (n = 3). *P < 0.01.

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  Figure 5. Contributions of MAPK/ERK signaling pathway in acute and persistent FMDV infection. The RNA expression levels of c-Raf and FMDV 3D in BHK-21 cells (A) and BHK-VEC cells (B) were verified by RT-qPCR. The protein expression levels of ERK and FMDV 3D and the phosphorylation of ERK in BHK-21 cells (C) and BHK-VEC cells (D) were verified by Western blot. The infectious FMDV titers in BHK-21 cells and BHK-VEC cells (E) were verified by TCID50 assay. OE, over expression; NC, negative control. GAPDH was served as the internal reference to normalize data. Three independent replicates were conducted for each sample. Data are expressed as the means ± SE (n = 3). *P < 0.01.

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  Figure 6. The effects of transcription factor FOS on acute and persistent FMDV infection. The RNA and protein expression levels of FOS in BHK-21 after transfected with shRNA were verified by RT-qPCR (A) and Western blot (B). The infectious FMDV titers in BHK-21 cells (C) and BHK-VEC cells (D) were verified by TCID50 assay. The RNA and protein expression levels of FOS and FMDV 3D in BHK-21 after downregulation of FOS in BHK-21 cells were verified by RT-qPCR (E) and Western blot (F). The RNA and protein expression levels of FOS and FMDV 3D in BHK-21 after upregulation of FOS in BHK-21 cells were verified by RT-qPCR (G) and Western blot (H). The RNA and protein expression levels of FOS and FMDV 3D in BHK-VEC after downregulation of FOS in BHK-VEC cells were verified by RT-qPCR (I) and Western blot (J). The RNA and protein expression levels of FOS and FMDV 3D in BHK-VEC after upregulation of FOS in BHK-VEC cells were verified by RT-qPCR (K) and Western blot (L). Sh, shRNA; OE, over expression; NC, negative control. GAPDH was served as the internal reference to normalize data. Three independent replicates were conducted for each sample. Data are expressed as the means ± SE (n = 3). *P < 0.01.

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  Figure 7. The schematic diagram depicting the regulation of MAPK/ERK signaling pathway on the evolution FMDV-persistent infection.