Fig. 8

Induction of TNF-α following neuronal ZIKV infection is a major regulatory mechanism that alters expression of genes relevant to neuronal function. A, B Expression values of candidate genes previously shown to be either downregulated (A) or upregulated (B) by ZIKV infection were measured via qRT-PCR in neuronal cultures treated for 24 h with 10 pg/ml exogenous cytokines: IFN-β, IL-6, or TNF-α. n = 6. C–E Volcano plots depicting mean z-score and -log10FDR of representative DEGs in neurons treated with IFN-β (C), IL-6 (D), or TNF-α (E). Significant differences (p < 0.05) are noted in red (upregulated DEGs) or blue (downregulated DEGs). n = 6. F, G) Concentrations of TNF-α in supernatants of in vitro neuronal cell cultures infected with ZIKV for 24 h (n = 4) (F) and brains harvested following in vivo intracranial infection (n = 5) (G). Cytokine concentrations were quantified via ELISA assay. H–M) qRT-PCR analysis was performed for known TNF-α transcriptional target genes, Cd69 (H), Ccl2 (I), Ccl5 (J), Stat1 (K), and TNF-α receptor genes, Tnfrsf1a (L) and Tnfrsf1b (M), in 8-week-old adult mouse brains following ZIKV-MR766 infection at 2 or 4 dpi. n = 5. N, O qRT-PCR analysis of representative DEGs associated with neurological functions (Chac1, Nr4a2, Lhx6, Dpp6, Cacna1e, and Tor3a) following pretreatment with neutralizing antibodies against IFN α/β receptor (IFNAR) (N) or TNFR1 (O) and subsequent 24 h infection with ZIKV-MR766. n = 3–6 biological replicates per group. Data in (A) and (B) represent normalized and z-transformed values of qRT-PCR expression data. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means