Department of Pediatrics, Xijing Hospital, Fourth Military Medical University, 15th Changlexi Road, Xi'an 710032, PR China.
J Biomed Sci. 2012 Aug 13;19(1):73. doi: 10.1186/1423-0127-19-73.
Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71.
Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2'-modified (2'-O-methylation or 2'-fluoro modification) siRNAs were designed to target highly conserved 5' untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs.
Transfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5' UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2'-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts.
Sequences were identified within the highly conserved 5' UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2'-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.
肠道病毒 71 型（EV71）是一种高度传染性病原体，在手足口病中起病因作用。它与严重的神经并发症有关，并在最近的大规模爆发中造成了重大死亡。目前，尚无针对 EV71 的有效疫苗或特定临床疗法。
设计了未经修饰的 21 个核苷酸小干扰 RNA（siRNA）和经典的 2'-修饰（2'-O-甲基化或 2'-氟修饰）siRNA，以靶向 EV71 基因组的高度保守 5'非翻译区（UTR），并用作抗 EV71 剂。实时 TaqMan RT-PCR、western blot 分析和蚀斑测定用于评估 siRNA 对特定病毒的抑制作用。
用靶向 EV71 基因组 5'UTR 的 siRNA 转染横纹肌肉瘤（RD）细胞，显著延迟和减轻 EV71 感染的细胞病变效应，增加 EV71 感染的 RD 细胞活力。对 EV71 复制的抑制作用具有序列特异性和剂量依赖性，病毒 RNA、VP1 蛋白和病毒滴度均有显著下降。与未经修饰的 siRNA 相比，适当的 2'-修饰 siRNA 具有相似的 RNA 干扰（RNAi）活性，并且血清稳定性显著提高。
在高度保守的 5'UTR 中鉴定出可通过 RNAi 策略有效抑制 EV71 复制的序列。适当的 2'-修饰 siRNA 为优化 siRNA 以克服基于 RNAi 的抗病毒治疗的广泛应用障碍提供了一种有前途的方法。
