Fig 1.
Comparative growth curves and normalized gene counts for Egyptian rousette bat (R06E) and Jamaican fruit bat (Aji) cell lines with African (MR766) and Asian-American (PRVABC59) ZIKV isolates.
(A) Growth curves of ZIKV/MR766 and PRVABC59 isolates in R06E cells over 72 hours post-inoculation (HPI). (B) Aji cells did not support viral growth for either ZIKV isolate. Dots represent individual replicates; the lines represent medians. Repeated measures ANOVA revealed significant differences between virus strains in R06E cells at 24, 48, and 72 HPI (*** p<0.0001), but no significant differences in Aji cells at any time point. (C) Spearman correlation analysis of normalized gene counts in R06E cells showed high similarity among samples, with a minimum coefficient of 0.9201. (D) Aji samples exhibited even higher similarity, with a minimum Spearman coefficient of 0.9625. The colored bar legend in the bottom right corresponds to all panels of the figure.
Fig 2.
Gene set enrichment analysis (GSEA) of R06E cells following ZIKV infection. The top 10 upregulated gene sets at 6 HPI (FDR < 0.1) are shown, along with normalized enrichment scores (NES) across all timepoints. Grey boxes indicate timepoints where no NES was calculated due to a lack of upregulated genes in the gene set.
(A) ZIKV/MR766-infected R06E cells showed enrichment of antiviral and pro-inflammatory gene sets. (B) ZIKV/PRVABC59-infected R06E cells displayed a similar enrichment pattern. (C) Direct comparison of ZIKV/MR766 vs. PRVABC59 in R06E cells revealed reduced enrichment of the same gene sets. (D) KEGG ID to associated pathway.
Fig 3.
Gene ontology enrichment analysis (GOEA) of differentially expressed genes (DEGs) in ZIKV-infected R06E cells. GOEA was performed on DEGs from each viral contrast to identify enriched GO terms. Terms were grouped by GO slim categories, with the most significant term representing each group. The top 10 enriched GO terms are shown, color-coded by unadjusted p-values.
(A) ZIKV/MR766-infected R06E cells showed strong enrichment for antiviral pathways. (B) ZIKV/PRVABC59-infected cells displayed a similar enrichment pattern. (C) Direct comparison of ZIKV/MR766 vs. PRVABC59 revealed reduced enrichment, with lower significance and fewer genes contributing to each GO term. (D) GO identification numbers to functional categories.
Fig 4.
Clustering of differentially expressed genes (DEGs) in R06E cells infected with African (MR766) and Asian-American (PRVABC59) ZIKV isolates. Boxes represent quartiles and median values, while dots indicate mean relative abundance per gene.
(A) Group 10 genes increased in expression over time, with larger divergence at 6 HPI. (B) GOEA revealed enrichment for defense response terms. (C, E) Groups 5 and 8 showed increased expression at 6 and 48 HPI in MR766-infected cells. (D, F) However, some enriched GO terms were not clearly linked to antiviral activity. For bars labelled with GO IDs, see S3 Table for full GO terms.
Fig 5.
Differential gene expression and network analysis of cytokine-related genes in ZIKV/MR766 vs. PRVABC59.
(A) Gene set enrichment analysis (GSEA) for “Viral protein interaction with cytokine and cytokine receptor” in ZIKV/MR766 vs. PRVABC59 (upregulated DEGs). The running enrichment score (ES) peaks early, driven primarily by CCL20, CCL5, CXCL8, and IL6, with a normalized enrichment score (NES) of 1.955—the highest in this contrast. (B) Log₂ fold changes of set genes show upregulation across HPI, with the leading genes among the most highly upregulated. (C) Protein-protein interaction (PPI) network from the STRING database, highlighting CCL5, CXCL8, and IL6 as key hub genes with extensive connectivity.
Fig 6.
Hypothesized mechanisms governing host-virus interactions during in vitro infection of Rousettus aegyptiacus cells with Marburg (MARV) and Zika (ZIKV).
((A) MARV infection in humans and non-human primates, (B) MARV infection in Rousettus aegyptiacus, (C) Zika virus infection in humans and non-human primates, and (D) Zika virus infection in Rousettus aegyptiacus. Created in BioRender by Fagre, A. (https://BioRender.com/04efbsj) and is licensed under CC BY 4.0.