ZIKV infection strongly induces antiviral immune responses in human NPCs. ZIKV infection strongly induces antiviral immune responses in human NPCs

To investigate the cellular pathologies in human NPCs after ZIKV infection, we performed global transcriptome profiling of the FD, FV, and H&S organoids at 3 and 6 dpi. We found 105 upregulated genes in the FD NPCs, 60 genes in the FV NPCs, and 168 genes in the H&S NPCs, with at least 1.5-fold differences found between ZIKV-infected groups and the control groups at 6 dpi. Among them, 28 genes overlapped in all three regional NPCs. These 28 common genes exclusively referred to the pathways associated with immune responses to viral infection, which could be clustered to the ISG family. Overall design: Three regional organoids were infected with ZIKV at MOI=0.5 or mock infection, and total RNAs were extracted at 3 dpi or 6 dpi and used for global transcriptome analysis. RNA-seq libraries were generated from duplicated samples per condition using the Next Illumina Ultra RNA library prep kit (NEB) following manufacturer’s protocols. RNA concentration of library was measured using Qubit RNA Assay Kit in Qubit 2.0. The insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and then accurate quantification was performed with Taqman fluorescence probe of AB Step One Plus Real-Time PCR system and sequenced by an Illumina Hiseq 2500 platform.

为探究寨卡病毒（Zika Virus, ZIKV）感染后人神经前体细胞（neural progenitor cells, NPCs）的细胞病理特征，我们对感染后第3天和第6天（days post infection, dpi）的额背侧（Frontal Dorsal, FD）、额腹侧（Frontal Ventral, FV）及海马-隔区（Hippocampal and Septal, H&S）类器官开展了全转录组分析。在感染后第6天，寨卡病毒感染组与对照组间存在至少1.5倍表达差异的上调基因分别为：FD神经前体细胞（FD NPCs）中105个、FV神经前体细胞（FV NPCs）中60个、H&S神经前体细胞（H&S NPCs）中168个。其中，28个基因在三类区域的神经前体细胞中均存在表达重叠。上述28个共有基因仅富集于抗病毒免疫应答相关通路，可归类为干扰素刺激基因（Interferon Stimulated Gene, ISG）家族。

实验整体设计如下：将三类区域类器官以感染复数（Multiplicity of Infection, MOI）=0.5的剂量感染ZIKV，同时设置模拟感染（mock infection）对照组；分别于感染后3天和6天提取总RNA，用于全转录组分析。每个实验条件设置生物学重复样本，采用Illumina Ultra RNA文库制备试剂盒（New England Biolabs, NEB）并依照厂商操作规程构建RNA-seq文库。使用Qubit 2.0荧光定量仪与Qubit RNA检测试剂盒测定文库浓度；通过安捷伦生物分析仪2100系统（Agilent Technologies, 美国加利福尼亚州）评估文库插入片段大小，随后采用AB Step One Plus实时荧光定量PCR系统的Taqman荧光探针进行准确定量，最终依托Illumina Hiseq 2500平台完成测序。