Oligonucleotides used for cloning.

Non-human primate (NHP)-based model systems are highly relevant for biomedical research. However, only few NHP cell lines are available and the generation of additional cell lines is an urgent need to help in the refinement and replacement of these models. Using lentiviral transduction of c-Fos, we established cell lines from the brain of rhesus macaques (Macaca mulatta). Transcriptome analysis revealed that these cell lines are closely related to astrocytes, which was confirmed by immunoblot and immunofluorescence microscopy detecting expression of the astrocyte marker glial fibrillary acidic protein (GFAP). Quantitative real-time PCR (qRT-PCR) demonstrated that major pathways of the interferon (IFN) system are intact. Using retroviral pseudotypes we found that the cell lines are susceptible to entry driven by the glycoproteins of vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and to a lesser extent influenza A virus (IAV). Finally, these cells supported growth of Zika virus (ZIKV) and Papiine alphaherpesvirus 2 (PaHV2). In summary, we developed IFN-responsive cell lines from the rhesus macaque brain that allowed entry driven by several viral glycoproteins and were permissive to infection with ZIKV and a primate simplexvirus. These cell lines will be useful for efforts to analyze neurotropic viral infections in rhesus macaque models.以非人灵长类（NHP）为基础的模型系统在生物医学研究中具有极高的应用价值。然而，当前可获取的非人灵长类细胞系数量极少，因此建立更多此类细胞系，对于推动这类模型的优化与替代工作具有迫切需求。本研究通过对c-Fos进行慢病毒转导，从恒河猴（Macaca mulatta）的脑组织中成功构建了细胞系。转录组分析结果显示，这些细胞系与星形胶质细胞具有高度同源性；免疫印迹与免疫荧光显微镜检测进一步证实，该细胞系可表达星形胶质细胞标志物胶质纤维酸性蛋白（GFAP），验证了上述结论。实时定量聚合酶链反应（qRT-PCR）实验表明，干扰素（IFN）系统的主要通路均保持完整功能。利用逆转录病毒假型系统进行的实验发现，这些细胞系对水疱性口炎病毒（VSV）、淋巴细胞脉络丛脑膜炎病毒（LCMV）的糖蛋白介导的病毒入侵具有易感性，同时对甲型流感病毒（IAV）的入侵敏感性相对较弱。此外，该细胞系可支持寨卡病毒（ZIKV）与狒狒α疱疹病毒2型（PaHV2）的增殖。综上，本研究成功构建了源自恒河猴脑组织的干扰素响应型细胞系，该细胞系可介导多种病毒糖蛋白驱动的病毒入侵过程，并可被寨卡病毒及灵长类单纯疱疹病毒感染。此类细胞系将为恒河猴模型中嗜神经性病毒感染的相关研究提供有力工具。