Fig. 5: Transcriptomic analysis of A549 cells transfected with VEEV replicase mRNA and SARS-CoV-2 mRNA (treatment group, n = 3) compared to cells transfected with an unrelated mRNA and SARS-CoV-2 mRNA (control group, n = 3).

A, B Volcano plots showing differentially expressed genes at 4 h (A) and 12 h (B) post-transfection. Cut-off values of log₂ fold change and p-value were set to ± 2.00 and 0.05. Significant downregulation of interferon-stimulated genes (ISGs) and other immune-related genes, such as IFNL2, IFNL3, IFNB1, CCL5, RAET1L, KRT17, and SEMA3D, were observed. C Venn diagram comparing the overlap of suppressed genes at 4 h and 12 h post-transfection, in the treatment group. A total of 20 genes were consistently downregulated at both time points, with 57 genes uniquely suppressed at 12 h and 28 genes at 4 h. D Combined dot and line plots demonstrating the expression of selected key ISGs (CCL5, IFNB1, IFNL1, IFNL2, IFNL3, IFNL4) over time, showing sustained suppression at 12 h post-transfection in the cells transfected with the treatment group (indicated by red lines) compared to the control group (blue lines). E, F Gene Ontology (GO) term enrichment analysis of suppressed genes at 4 h (E) and 12 h (F). Pathways related to immune response remained consistently suppressed at both time points. At 12 h, significant suppression was observed in crucial pathways involved in anti-viral responses, including type I interferon signaling, serine phosphorylation of STAT proteins, and cytokine-mediated signaling. The size of the circles represents the gene ratio within each GO term, and the color gradient corresponds to the adjusted P-value. These findings highlight the temporal dynamics of immune response modulation following transfection of epithelial cells with the VEEV replicase mRNA and SARS-CoV-2 mRNA (treatment group), emphasizing the plausible role of VEEV replicase in the prolonged suppression of critical antiviral pathways at later time points.