Diversities of African swine fever virus host-virus dynamics revealed by single-cell profiling

Figures

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  Fig 1. Landscape of single-cell transcriptome dynamics in PAMs exposed to ASFV. (A) Immunofluorescence of ASFV viral protein p30 in cells exposed to different ASFV strains at different hpi. Scale bar = 100 µm. (B) Bar plots showing the proportion of p30 positive cells for each ASFV strain at the different hpi. Data are shown as mean value ± SD, n = 3. (C) Schema of this study design. (D) UMAP plots showing the cell types in the integrated single-cell transcriptomes of 51,946 cells derived from ASFV unexposed and exposed cells. (E) Dot plot showing scaled expression levels of cell type–specific genes in unexposed and exposed cells. Expression levels in each gene are scaled, ranging from 0 to 1. The dot color and size indicate expression levels and the percentage of expressed cells, respectively. DC, dendritic cell. (F) UMAP plots showing the distribution of unexposed cells and exposed cells.

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  Fig 2. Attenuated and low-virulence ASFV strains tend to exhibit higher viral load compared with highly virulent ASFV strain. (A) Line plots showing the expression levels of representative viral genes. Gene expression levels are quantified with counts per million mapped reads (CPM). Data are shown as mean value ± SEM. (B) UMAP plot showing the normalized viral load in cells in each virus strain. 0, 06, 12, 24, and 48 mean hours post-infection (hpi). (C) Box and point plot showing the viral load in each sample. Data are shown as mean ± SEM. (D) PCA plot showing the distribution of each sample by the proportional viral gene expression. (E) Heatmap plot showing expression levels of ASFV genes in each sample. (F) Line plots showing average expression levels of viral genes in four gene clusters. The number of viral genes in each gene cluster is indicated in parentheses. (G) Brief schematic overview of ASFV RNA polymerase to induce ASFV transcription. (H) Bar plots showing qRT-PCR results of ASFV RNA polymerase subunits genes transcription. ns, not significant; **P < 0.01 and ***P < 0.001 (two-tailed t test). Data are shown as mean value ±SD, n = 3.

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  Fig 3. The interferon pathways were activated in PAMs exposed to attenuated and low virulent ASFV. (A) Dot plot showing the DEGs between unexposed and exposed cells. Each dot corresponds to a gene (red means upregulated and blue means downregulated). (B) Bar plots showing enriched GO terms of upregulated genes in exposed cells. (C) Bar plots showing GO terms of downregulated genes in exposed cells. (D) UMAP plot showing the distinct composition of cells without or with exposed to three ASFV strains (exposed to SY18, HuB20, and I226R). The different colors indicated different hpi. (E) PCA plot showing the distribution of each sample by the host gene expression. (F) Heatmap depicting the expression levels of top 10 genes by log2FC enriched in each ASFV strains exposed PAMs at 48hpi. ISG genes are highlighted using green color. (G) Bar plots showing the number of DEGs among three ASFV strains exposed PAMs at 48 hpi. (H) Dot plots showing enriched GO terms of genes highly expressed in SY18 or I226R exposed cells at 48 hpi. (I) Violin plots showing ISG score among three ASFV strains exposed PAMs at 48 hpi. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed t test). (J) Line plot showing ISG…

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  Fig 4. The interferon pathways were activated through the IRF7-mediated positive feedback loop in PAMs exposed to attenuated and low virulent ASFV. (A) Dot plots showing potential key regulators in cells exposed to three ASFV strains. The dot color and size indicate ASFV strains and the number of connections. (B) Brief schematic overview of virus induced IFN activation pathway. (C) The violin plots show the expression levels of IRF3 and IRF7 among three ASFV strains exposed PAMs. (D) Scatter plots showing the correlation of normalized expression levels of IRF3 and IRF7 (horizontal axis) with ISG score (vertical axis) in cells exposed to three ASFV strains. (E) Bar plots showing the qRT-PCR results of the relative transcriptional level of IRF3, IRF7, IFI6, and IFIT1 in cells exposed to different ASFV strains. ns, not significant; *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed t test). Data are shown as mean value ± SD, n = 3.

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  Fig 5. Subclustering of PAMs revealed specific populations that regulate host response to different virulent ASFV strains. (A) UMAP plots showing nine populations of PAMs among unexposed and exposed cells at each hpi. (B) Dot plot showing expression patterns of selected genes across indicated PAMs populations. (C) Heatmap showing the sample preference of each PAMs population estimated by Ro/e. *R O/e>1, ** R O/e > 5 and *** R O/e > 10. (D) Violin plots showing ISG and inflammatory score for each PAMs population. (E) Violin plots showing the expression levels of SIGLEC1, CD163, and IL18 across different PAMs populations. (F) UMAP plot showing expression levels of representative genes and corresponding PAMs populations are indicated with a blue circle. (G) Bar plots illustrating the transcription levels of CD163, SIGLEC1 and IL18 in PAMs exposed to three ASFV strains at 48 hpi using qRT-PCR. ns, not significant; **P < 0.01 and ***P < 0.001 (two-tailed t test). Data are shown as mean value ± SD, n = 3.

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  Fig 6. Association analysis between viral load and host genes identifies CEBPB as a target gene negatively regulated by ASFV replication. (A) Volcano plot showing the association between host gene expression and viral load within three ASFV strains exposed PAMs. The downregulated gene number refers to the number of host genes that were significantly negatively correlated with viral load, whereas the upregulated gene number refers to the number of host genes that were significantly positively correlated with viral load. (B) Boxplot showing the number of genes upregulated (positively correlated with viral load) or downregulated (negatively correlated with viral load) in PAMs exposed to three ASFV strains. (C) Dot plots showing GO terms of upregulated genes responded to increasing viral load in SY18, HuB20, and I226R. (D) Dot plots showing GO terms of downregulated genes responded to increasing viral load in SY18, HuB20, and I226R. (E) Bar plots showing the qRT-PCR results of relative transcriptional level of CEBPB in cells exposed to different ASFV strains at 6, 12, 24, and 48hpi. Data are shown as mean values ± SD, n = 3. (F) Western blot experiment showing results of CEBPB protein level…

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  Fig 7. Graphical summary of the differences in PAM cells exposed to ASFV strains of varying virulence. Schematic overview of the work: (1) attenuated and low virulent ASFV strains, HuB20 and I226R, exhibited higher viral load compared with the highly virulent strain SY18; (2) CEBPB was identified as a target gene negatively regulated by ASFV viral load in all three AFSV strains; (3) IRF7-mediated interferon positive feedback loop pathway contributed to the promotion of ISGs transcription in cells exposed to HuB20 and I226R, but not SY18; and (4) pro-inflammatory cytokine IL18 was highly expressed in cells exposed to SY18 compared to cells exposed to HuB20 and I226R at 48 hpi.