Supplement 3.xlsx

Enterovirus D68 (EV-D68) causes a diverse range of infectious diseases, especially in children. Until 2010, EV-D68 was primarily associated with mild respiratory diseases. However, since 2010, it has been identified as a cause of severe respiratory illness, requiring hospitalization and intensive care unit admission. The increasing number of acute flaccid paralysis cases linked to EV-D68 infection has become a global public health concern since 2014. No effective vaccines or drugs are currently available for treatment. Through drug screening, we discovered that Geranyl-<i>p-trans-</i>coumaric acid(GCA), a pure compound extracted from Taiwanese propolis, could be a potential inhibitor against EV-D68. The underlying molecular mechanism was determined through a series of <i>in vitro</i> assays. We further confirmed this through drug-resistant virus generation and drug-protein interaction using molecular docking. As a result, GCA directly bound to viral capsid in VP1 at the canyon and heparan sulfate proteoglycan binding sites. This binding did not interfere with viral attachment to the cell surface but specifically inhibited viral uncoating. A single mutation at VP1 T92N was found to affect the binding site, however, it did not confer resistance to GCA. Additionally, we propose that unidentified cellular host factors may be involved in EV-D68 entry, potentially associated with the nonstructural viral protein 2C. Notably, only double mutations (VP1 T92N and 2C K6R) conferred resistance to GCA, suggesting a partial but cooperative role in the antiviral mechanism. The antiviral efficacy of GCA was further confirmed in an animal model. Collectively, our study not only identifies a promising inhibitor but also provides novel mechanistic insights into the viral entry process in EV-D68 life cycle.

肠道病毒D68（Enterovirus D68, EV-D68）可引发多种感染性疾病，尤其多见于儿童。2010年之前，EV-D68主要与轻度呼吸道疾病相关。然而自2010年起，该病毒被证实可导致严重呼吸道疾病，患者需住院治疗甚至进入重症监护室（intensive care unit, ICU）。2014年以来，与EV-D68感染相关的急性弛缓性麻痹（acute flaccid paralysis, AFP）病例数持续增加，已成为全球公共卫生关注的焦点。目前尚无有效的疫苗或药物用于治疗。通过药物筛选，我们发现从台湾蜂胶中提取的纯化合物香叶基-对反式香豆酸（Geranyl-p-trans-coumaric acid, GCA）可能是EV-D68的潜在抑制剂。其潜在分子机制通过一系列体外实验（in vitro assays）得以阐明。我们通过耐药病毒生成及分子对接（molecular docking）技术研究药物-蛋白相互作用，进一步验证了这一机制。结果显示，GCA直接结合于病毒衣壳（viral capsid）VP1蛋白的峡谷区及硫酸乙酰肝素蛋白聚糖（heparan sulfate proteoglycan）结合位点。这种结合不干扰病毒与细胞表面的附着，但能特异性抑制病毒脱壳（viral uncoating）。研究发现VP1蛋白T92N位点的单一突变会影响结合位点，但并未使病毒获得对GCA的耐药性。此外，我们推测未被鉴定的细胞宿主因子（cellular host factors）可能参与EV-D68的入侵过程，且可能与病毒非结构蛋白2C相关。值得注意的是，仅VP1 T92N与2C K6R的双重突变可使病毒对GCA产生耐药性，这表明两者在抗病毒机制中发挥部分协同作用。GCA的抗病毒效果在动物模型中得到进一步验证。综上，本研究不仅发现了一种具有潜力的抑制剂，还为EV-D68生命周期中的病毒入侵过程提供了新的机制见解。