Fig. 3: Global transcriptional response in human and bat cells upon IFNβ stimulation.

P. alecto (PaKiT03), E. fuscus (Efk3B), and human (A549, RTPEC) cells were untreated or treated with 1 U/mL of species-matched IFNβ for 6 h. A Schematic of sample preparation for transcriptomics using bulk RNA sequencing. Image created in BioRender [https://BioRender.com/o9oes1x]. B Principal component analysis (PCA) depicting global transcriptional profiles of the IFNβ-stimulated samples. C Enriched GO terms for IFNβ-treated cells. Dot color represents fold enrichment, and size represents −log₁₀ values. A hypergeometric test (cumulative distribution function; upper tail) with a subsequent multiple hypothesis Benjamini & Hochberg correction of the frequency of terms associated with genes in the significantly differentially expressed gene set compared to a genome background was used to determine a p value for over enrichment. D Volcano plots depicting DEGs in A549, RPTEC, PaKiT03, and Efk3B cells. DEGs (p adjusted <0.05) with a log₂ fold change of more than 2 are indicated in red. Non-significant DEGs with a fold change of less than 2 are indicated in green. Statistical test and multiple hypothesis correction was performed using DESeq2 to identify DEGs. Bat and human icon were obtained from BioRender. E Heatmap indicating the expression levels of the top DEGs in bats ranked by p value involved in the IFNβ response. The ranking p value comes from the DESeq2 DESeq function analysis of bat gene expression. E. fuscus transcript paralogs are indicated in brackets (i. e., Par.1) and are plotted against the transcript levels of a single variant detected in PaKiT03 and RPTEC cells. See Supplementary Data 4 for the representative LOC symbols. F Luciferase activity from rabbit reticulocyte lysate incubated with cap0 or cap1-β-globin-Fluc RNA, Zika virus Fluc RNA, or SARS2-Fluc RNA in the presence of human or P. alecto IFIT1. Data were normalized to the luciferase activity in the absence of IFITs and are shown as the mean ± the standard error of three separate experiments (Two-way ANOVA, Tukey’s range test). G P. alecto IFIT1 and IFIT3 were expressed and purified from E. coli and analysed by SEC as indicated. Peak fractions from the IFIT1 + IFIT3 (complex) run analysed by SDS-PAGE are shown below the elution trace. H Mutated P. alecto IFIT1 and wildtype IFIT3 were expressed and purified from E. coli and analysed by SEC as indicated. Peak fractions from the IFIT1 + IFIT3 (complex) run analysed by SDS-PAGE are shown below the elution trace. I Transcripts per million values for GBP1 across PaKiT03, EfK3B, A549, and RTPEC cells. Data were representative of three biological replicates.