Time-Resolved Transcriptomic Profiling of Chandipura Virus Infection Reveals Dynamic Host Responses and Host-Directed Therapeutic Targets

Figures

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  Figure 1. Microscopic images of HEK293T cells infected with Chandipura virus at different time points. Representative bright-field images of HEK293T cells at 0, 6, 12, 18, and 24 h after infection (hpi). The top row shows uninfected control cells at corresponding time points. The bottom row shows infected cells, demonstrating time-dependent cytopathic effects, including cell rounding and detachment. Scale bar: 200 μm.

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  Figure 2. Transcriptome analysis of differentially expressed genes (DEGs) across time points after infection. (a) Principal Component Analysis (PCA) plot showing clustering of samples (represented by dotted lines) based on gene expression profiles at each time point (0, 6, 12, 18, and 24 hpi), indicating time-dependent transcriptomic shifts. (b) Bar plots depicting the number of significantly upregulated and downregulated genes (adjusted p-value < 0.05, |log2FC| ± 1) for each time point compared to 0 hpi. (c) Upset plots showing the number of unique and overlapping upregulated (left) and downregulated (right) DEGs across different time points.

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  Figure 3. Differential expression profiles of host genes in response to Chandipura virus infection. (a) Volcano plots showing the distribution of DEGs (red: upregulated, green: downregulated) at 6, 12, 18 and 24 hpi versus control (0 hpi). Colored dots represent significant genes (adjusted p-value < 0.05, |log2FC| ± 1). (b) Heatmap showing the top 30 most variable genes across samples. Gene expression counts were normalized using variance stabilizing transformation (DESeq2) and mean-centered per gene. Columns represent biological replicates at each time point (0, 6, 12, 18, and 24 hpi). Red indicates higher expression and blue indicates lower expression relative to the gene’s average across samples. Genes were hierarchically clustered.

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  Figure 4. Biological pathway enrichment of differentially expressed genes across time points. Dot plots representing enriched Gene Ontology (GO) biological processes for DEGs at 6 hpi vs. 0 hpi (a), 12 hpi vs. 0 hpi (b), 18 hpi vs. 0 hpi (c), and 24 hpi vs. 0 hpi (d). The size of the dots indicates the number of genes associated with each term, while color intensity corresponds to the statistical significance (−log10 adjusted p-value).

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  Figure 5. KEGG pathway enrichment analysis of differentially expressed genes at multiple time points following Chandipura virus infection. Dendrograms show clustering of significantly enriched KEGG pathways based on differentially expressed genes (DEGs) at 6 hpi vs. 0 hpi (a), 12 hpi vs. 0 hpi (b), 18 hpi vs. 0 hpi (c), and 24 hpi vs. 0 hpi (d). Pathways are color-coded based on enrichment direction: green nodes represent significantly downregulated pathways, and red nodes represent significantly upregulated pathways (adjusted p-value < 0.05).

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  Figure 6. In silico identification of potential drug candidates. Drug–gene interaction network generated using the Network Analyst tool for (a) SLC7A11 and (b) HDAC4. The target gene is shown as a green node, while interacting drugs/metabolites are shown as blue nodes. Sulfasalazine and romidepsin (highlighted with a red box) were selected based on published literature. (c) Predicted protein–ligand interaction map of Nilotinib within the binding pocket of the EGFR1 during the induced fir docking, illustrating key interacting residues and non-covalent interactions that support the network-based prediction.

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  Figure 7. Microscopic images of in vitro analysis of Sulfasalazine, Romidepsin, and Nilotinib drugs in SHSY5Y cells against CHPV infection. (a) The top row shows the uninfected control cells and the bottom row shows the CHPV-infected cells at 0 hpi, 24 hpi, and 48 hpi. (b) Microscopic images of SHSY5Y cells, treated with different concentrations (12.5 µM, 25 µM, 50 µM, and 100 µM) of Sulfasalazine drug at 0, 24, and 48 h after CHPV infection. (c) Microscopic images of cells treated with Romidepsin drug at 5 nM, 10 nM, and 20 nM at 0, 24, and 48 hpi. (d) Microscopic images of cells treated with Nilotinib drug at 1.25 µM, 2.5 µM, 5 µM, and 10 µM concentrations. Scale bar: 200 µm.

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  Figure 8. Antiviral activity and cytotoxicity profiling of Sulfasalazine, Romidepsin, and Nilotinib drugs against CHPV. CHPV-infected SHSY5Y cells were treated with increasing concentrations of sulfasalazine (a), romidepsin (b), or Nilotinib (c) and its effect on viral replication was assessed by RT-qPCR. The left y-axis shows the ∆CT values for the N2, N3, and P viral genes, where higher ∆CT values indicate reduced viral load. ∆CT was calculated by subtracting CT value of CHPV-infected cells from the CT value of CHPV-infected, drug-treated cells. Cell viability (%), measured at 48 h after drug treatment using the MTT assay, is shown on the right y-axis.