The increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. Serum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. Compared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment. Our results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

中文翻译：

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易位脂多糖通过促进促炎症和病毒 IRES 活性与肠道病毒 A71 诱导的手足口病的严重程度相关

最近在由肠道病毒 A71 (EV-A71) 引起的严重手足口病 (HFMD) 中观察到引起炎症的肠杆菌增加。本研究旨在验证细菌易位 (BT) 的发生，并进一步探讨 BT 对 EV-A71 介导的手足口病病例严重程度的影响。收集了 65 名轻度和 65 名重度 EV-A71 相关手足口病病例和 65 名健康儿童的血清标本。血清中的EV-A71 VP1，炎症介质包括C-反应蛋白、IL-1β、IL-6、干扰素-γ和肿瘤坏死因子-α，BT相关生物标志物包括Claudin-3、肠脂肪酸结合蛋白、脂多糖（LPS） )、可溶性 CD14 (sCD14) 和内毒素核心抗体通过 ELISA 测量。细菌 DNA (BactDNA) 片段通过定量 PCR (qPCR) 进行定量。横纹肌肉瘤 (RD) 或 SH-SY5Y 细胞，用 LPS 预孵育的 EV-A71 感染或用含有病毒 2Apro 的质粒或含有病毒内部核糖体进入位点 (IRES) 的 mRNA 转染的小鼠，在体外用或不用 LPS 进行后处理。EV-A71 RNA 和病毒或细胞蛋白分别通过 qPCR 和蛋白质印迹测定。与轻度手足口病患者相比，在重度手足口病患者中观察到显着更高的炎症介质以及除 BactDNA 外的 BT 相关生物标志物（均 P < 0.05）。在重度手足口病组中，LPS和sCD14的循环浓度与炎症指标呈统计学相关（均P < 0.05），血清EV-A71 VP1水平与血清​​LPS呈正相关（r = 0.341，P = 0.005）和血清 sCD14（r = 0.458，P < 0.001）。体外，LPS 预孵育仅略微促进了 EV-A71 的附着和内化；然而，LPS 后处理显着加速了 EV-A71 增殖和病毒 2Apro 介导的 IRES 活性。我们的结果共同表明，肠道来源的易位 LPS 通过病毒 2Apro 介导的 IRES 驱动炎症反应和病毒增殖，从而导致 EV-A71 诱导的手足口病的严重程度。