Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.IMPORTANCE Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies.

中文翻译：

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当代循环肠道病毒D68株已获得了在人类神经元细胞中病毒进入和复制的能力。

肠道病毒D68（EV-D68）历来与呼吸系统疾病有关。然而，在2014年和2016年夏季，EV-D68爆发与脊髓灰质炎样急性弛缓性脊髓炎/麻痹（AFM / AFP）病例激增同时发生。这引起了人们的担忧，即在最近的爆发中，EV-D68可能是AFM的病原体。为了评估EV-D68的潜在神经向性，我们利用源自神经母细胞瘤的神经元细胞系SH-SY5Y作为细胞培养模型来确定是否观察到针对不同EV-D68菌株的差异感染。与支持所有受测EV-D68菌株的病毒感染的HeLa和A549细胞相反，SH-SY5Y细胞仅支持一部分当代EV-D68菌株的感染，包括2014年爆发的分离株。使用多种试验评估了SH-SY5Y中的病毒复制和感染性：病毒产生，细胞病变效应，细胞ATP释放和VP1衣壳蛋白产生。在分化的SH-SY5Y细胞，原代人神经元培养物和小鼠麻痹模型中也观察到了类似的差异性向神经性。使用SH-SY5Y细胞培养模型，我们确定病毒结合和进入的障碍至少部分负责差异感染性表型。将基因组RNA转染到所有EV-D68分离株的SH-SY5Y产生的病毒体中，但是从不能直接感染SH-SY5Y的菌株中仅观察到单轮复制。除了支持病毒复制和其他功能研究之外，这种细胞培养模型可能有助于鉴定毒力的特征，以确认EV-D68菌株与AFM之间的流行病学关联，并允许快速鉴定和表征新兴的神经营养菌株。自2014年夏季EV-D68爆发以来，有证据表明与肢体麻痹（AFM）类型的因果关系已经建立。在本文中，我们描述了一个神经元细胞培养模型（SH-SY5Y细胞），其中当代2014年暴发性EV-D68菌株的子集显示出神经元细胞的感染性或嗜神经性。我们证实了在体外使用原代人神经元细胞培养物和在体内使用小鼠麻痹模型的嗜神经性的差异。使用SH-SY5Y细胞模型，我们确定病毒进入的障碍至少部分负责神经嗜性。SH-SY5Y细胞可用于确定特定的EV-D68遗传决定因素是否与神经发病相关，并且该细胞系中的复制可用作鉴定嗜神经性EV-D68菌株的快速筛选工具。这可能有助于更好地了解发病机理和流行病学，并有助于开发潜在的疗法。