Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells.
Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells.
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DOI：
10.1128/mbio.01954-18
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发表时间：
2018-10-16
期刊：
影响因子：
6.4
通讯作者：
Scheuermann RH
中科院分区：
文献类型：
--
作者：
Brown DM;Hixon AM;Oldfield LM;Zhang Y;Novotny M;Wang W;Das SR;Shabman RS;Tyler KL;Scheuermann RH
自 2014 年夏季 EV-D68 爆发以来，越来越多的证据表明与一种肢体麻痹 (AFM) 存在因果关系。在本文中，我们描述了一种神经元细胞培养模型（SH-SY5Y 细胞），其中当代 2014 年爆发的 EV-D68 菌株的一个子集显示出神经元细胞的感染性或向神经性。我们在体外使用原代人类神经元细胞培养物和在体内使用小鼠麻痹模型证实了向神经性的差异。使用 SH-SY5Y 细胞模型，我们确定病毒进入的障碍至少部分是造成向神经性的原因。 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机制相关，并且该细胞系中的复制可用作鉴定神经亲性 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制和流行病学以及潜在疗法的开发。肠道病毒 D68 (EV-D68) 历来与呼吸道疾病有关。然而，在 2014 年和 2016 年夏季，EV-D68 爆发恰逢脊髓灰质炎样急性弛缓性脊髓炎/麻痹 (AFM/AFP) 病例激增。这引起了人们的担忧，即 EV-D68 可能是最近爆发的 AFM 的病原体。为了评估 EV-D68 的潜在向神经性，我们利用神经母细胞瘤来源的神经元细胞系 SH-SY5Y 作为细胞培养模型，以确定不同 EV-D68 株是否观察到差异感染。 HeLa 和 A549 细胞支持所有测试的 EV-D68 毒株的病毒感染，与此相反，SH-SY5Y 细胞仅支持当代 EV-D68 毒株的子集的感染，包括 2014 年爆发的分离株。使用多种测定法评估 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、原代人类神经元培养物和小鼠麻痹模型中也观察到了类似的差异神经向性。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在一定程度上造成了不同的感染性表型。将基因组 RNA 转染至 SH-SY5Y 中，产生所有 EV-D68 分离株的病毒颗粒，但在不能直接感染 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，该细胞培养模型可能有助于识别毒力特征，以确认 EV-D68 毒株和 AFM 之间的流行病学关联，并允许快速识别和表征新兴的嗜神经毒株。
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. 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.