Data_Sheet_1_Chikungunya virus infection induces ultrastructural changes and impaired neuronal differentiation of human neurospheres.PDF

Chikungunya virus (CHIKV) is an arthropod-borne virus recently associated with large outbreaks in many parts of the world. Infection is typically manifested as a febrile and self-limited illness, characterized by joint pain and myalgia, albeit severe neurological manifestations are also reported. Although CHIKV is not recognized as a truly neurotropic virus, neurons, astrocytes, and oligodendrocytes are susceptible to infection in vitro. Here we employed a model of 3D cell culture to obtain neurospheres from ATRA/BNDF differentiated human neuroblastoma cells. We demonstrate that CHIKV is able to establish a productive infection, resulting in ultrastructural changes in cell morphology and impaired neuronal differentiation. Ultrastructural analysis of neurospheres infected with CHIKV during neuronal differentiation revealed diminished neuron dendrite formation, accumulation of viral particles associated with the plasma membrane, numerous cell vacuoles, and swollen mitochondria. Apoptotic cells were significantly increased at 72 h post-infection. Compared to Zika virus, a well-characterized neurotropic arbovirus, CHIKV infection resulted in a more discrete, albeit detectable upregulation of IL-6 levels. Finally, we found that CHIKV infection resulted in an altered profile expression, mainly downregulation, of a group of transcription factors named Hox genes. Altogether our findings highlight important features of CHIKV in the CNS, as well as the feasibility of neurospheres as robust experimental models that can support further studies for novel pharmacological interventions.

基孔肯雅病毒（Chikungunya virus, CHIKV）是一类近年在全球多个地区引发大规模暴发的节肢动物传播病毒。感染后通常表现为发热性自限性疾病，以关节痛与肌痛为典型临床特征，但亦有重症神经系统表现的病例报道。尽管CHIKV尚未被归类为典型嗜神经性病毒，但体外实验证实神经元、星形胶质细胞与少突胶质细胞均对其易感。本研究采用三维细胞培养模型，从经全反式维甲酸（ATRA）/脑源性神经营养因子（BNDF）诱导分化的人神经母细胞瘤细胞中分离制备神经球。实验结果表明，CHIKV可在神经球中建立增殖性感染，引发细胞形态的超微结构改变，并损害神经元分化进程。对感染CHIKV的分化期神经球开展超微结构分析可见：神经元树突形成减少、病毒颗粒在质膜区域聚集、大量细胞空泡形成以及线粒体肿胀。感染后72小时，凋亡细胞数量显著升高。与特征明确的嗜神经性虫媒病毒寨卡病毒（Zika virus）相比，CHIKV感染引发的白细胞介素6（IL-6）水平上调更为隐匿，但仍可被检测到。此外，本研究发现CHIKV感染会改变一类名为同源盒基因（Hox genes）的转录因子的表达谱，主要表现为基因表达下调。综上，本研究结果揭示了CHIKV在中枢神经系统（Central Nervous System, CNS）中的重要致病特征，同时验证了神经球作为可靠实验模型的可行性，可为后续新型药物干预相关研究提供坚实基础。