Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro. Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates.

中文翻译：

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神经母细胞瘤细胞中增强的肠道病毒 D68 复制与 VP1 中的细胞培养适应性氨基酸替代相关

自 2014 年在美国出现以来，肠道病毒 D68 (EV-D68) 一直并且与严重的呼吸道疾病和急性弛缓性脊髓炎有关。尽管 EV-D68 已被证明可以在体外的不同神经元细胞中复制，目前尚不清楚哪些病毒因子有助于在中枢神经系统细胞中有效复制的能力，以及该特征是否是进化枝特异性特征。在这里，我们确定了来自（子）进化枝 A、B1、B2、B3 和 D1 的临床 EV-D68 分离株在人神经母细胞瘤细胞 (SK-N-SH) 中的复制动力学。随后，我们比较了序列以鉴定与病毒复制增加相关的病毒因子。所有临床分离株都在 SK-N-SH 细胞中复制，尽管效率存在很大差异。临床分离株的有效复制与 VP1 (E271K) 的 271 位氨基酸取代有关，这是在体外病毒繁殖过程中获得的. 除唾液酸外，硫酸乙酰肝素的识别与附着、感染和复制的增加有关。去除硫酸乙酰肝素导致 E271K 病毒的附着、内化和复制减少。总之，我们的研究表明 EV-D68 分离株在 SK-N-SH 细胞中的复制动力学不是进化枝特异性特征。然而，将硫酸乙酰肝素识别为一种额外的受体对体外表型特征有很大影响。这些观察结果强调需要将病毒原种的序列与临床分离株进行比较，以便从原始病毒分离株中检索表型特征。