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Figure 4 ZIKV Infection Causes Global Deregulation of Gene Expression Show full caption (A and B) RNA-seq analysis of whole ZIKV-infected and mock-infected littermate brains (E13.5–E16.5). Genes with significant differences in expression were subjected to GO analyses. The typical 20 most significant terms are selected for upregulated (A) and downregulated (B) genes, respectively. The −log10 p values are indicated by bar plots. (C and D) RNA expression analysis of RNA-seq results in Table S2 . Bar plots indicate induction of antiviral response genes (C) or the candidate flavivirus entry receptors (D) in the ZIKV-infected brains. (E) Expression of microcephaly-associated genes between viral injection side of brains and controls as determined by real-time PCR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001. See also All data are means ± SD.p < 0.05,p < 0.01,p < 0.005,p < 0.0001. See also Figure S4 and Table S2

To investigate the global impact of ZIKV infection on the whole developing brain at the molecular level, we carried out global transcriptome analyses (RNA-seq). Genome-wide analyses identified a large number of differentially expressed genes at 3 days after viral infection ( Table S2 ). Gene Ontology analyses revealed a particular enrichment of upregulated genes in immune-response-related and apoptosis pathways ( Figure 4 A and Table S2 ). Very notable were the genes related to cytokine production and the response to cytokines, suggesting that cytokines play a critical role in the pathogenesis of ZIKV infection ( Figure 4 A and Table S2 ). Many antiviral response genes were reported to be induced by ZIKV in human skin fibroblasts (). Although there is a much longer list of related genes in our dataset, we have confirmed all of those reported by Hamel et al., including Tlr3, Ddx58, Ifih1, Oas2, Isg15, and Mx1, in addition to Ccl5, Cxcl10, and Ifnb1, which were not detected in controls but were expressed in infected brains ( Figure 4 C and Table S2 ). Interestingly, many candidate flavivirus entry receptors were also induced, most notably AXL, which has been predicted as a ZIKV receptor () ( Figure 4 D and Table S2 ). In contrast, many genes involved in cell proliferation, differentiation, migration, and organ development were downregulated ( Figure 4 B and Table S2 ). It was of interest that most of the microcephaly-associated genes were significantly downregulated ( Figure S4 ) in the dataset of ZIKV-infected hNPCs (). We confirmed the significant downregulation of seven of them, including ASPM, CASC5, CENPF, MCPH1, RBBP8, STIL, and TBR2, in our dataset or by real-time PCR of the virus-injected sides of the brains ( Figure 4 E). The downregulation of microcephaly-associated genes that we found largely overlaps with that found in the hNPC dataset, although the downregulation of CEP152 and WDR62 was not very significant. Therefore, these global transcriptome datasets not only support our cell biology findings but also provide a useful resource for the exploration of the underlying pathogenesis and for the potential treatment of ZIKV infection when combined with a dataset from hiPSCs ().