Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium
Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium
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DOI：
10.1093/jac/dky089
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发表时间：
2018-07-01
影响因子：
5.2
通讯作者：
Jochmans, Dirk
中科院分区：
文献类型：
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作者：
Mirabelli, Carmen;Jaspers, Martine;Jochmans, Dirk
目的：我们报告了在气-液界面中使用支气管来源的重建的3D人气道上皮细胞（HuAEC）来研究呼吸道合胞病毒（RSV）感染并评估RSV抑制剂在（前）临床开发中的功效。用RSV-A Long株感染HuAEC（0.01 CCID 50/细胞，其中CCID 50代表HEp 2细胞中50%细胞培养感染剂量）。在感染时或感染后1天或3天，在培养物的基底隔室上添加所选抑制剂并每日更新。通过每天收集的顶端洗涤物和通过RT-qPCR.Results定量病毒RNA来跟踪病毒脱落。RSV感染在感染后4天降低了纤毛细胞的纤毛搏动频率，在第10天观察到完全的纤毛运动障碍。RSV融合抑制剂治疗仅在感染时加入时才产生抗病毒作用。与此相反，使用复制抑制剂（核苷和非核苷）引起了显着的抗病毒效果，即使治疗的开始被推迟到感染后1天，甚至3天。炎症标志物RANTES（mRNA）的水平在感染的未处理的培养物中增加了200倍（在感染后3周），但在存在RSV复制抑制剂PC 786的情况下，水平与未感染培养物的水平相当，这表明有效的抗病毒治疗可能抑制该模型中病毒诱导的炎症。总的来说，HuAEC提供了一个强大的和生理相关的模型来研究RSV复制和评估抗病毒化合物的疗效。
Objectives: We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-) clinical development.Methods: HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR.Results: RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleoside) elicited a marked antiviral effect even when the start of treatment was delayed until 1 day or even 3 days after infection. Levels of the inflammation marker RANTES (mRNA) increased similar to 200-fold in infected, untreated cultures (at 3 weeks post-infection), but levels were comparable to those of uninfected cultures in the presence of PC786, an RSV replication inhibitor, suggesting that an efficient antiviral treatment might inhibit virus-induced inflammation in this model.Conclusions: Overall, HuAECs offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.