腺病毒介导的miRNA干扰策略抗MERS-CoV的研究

In 2012, a novel coronavirus, termed MERS-CoV, emerged in the Middle East, and the developing trend has been worsening. To date, 184 human cases of MERS-CoV have been reported with 80 fatalities. Facing the increasing severity of epidemic, it is of utmost importance to develop a safe and effective vaccine against these newly emerging virus. RNA interference (RNAi) refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this regard, RNAi holds great promise as a novel nucleic acid–based therapeutic against MERS-CoV. In this study, we aimed to develop a novel anti-MERS therapeutic strategies based on RNAi and adenovirus delivery system. We designed and synthesized several artificial mricroRNAs (amiRNAs) complementary to MERS-CoV S and N genes respectively, then cloned these amiRNAs into a commercially available pcDNATM6.2-GW/EmGFP-miR vector. Vero cells were transfected with these plasmids, and then infected with MERS-CoV 24 hours later after transfection, screening for the amiRNAs of inhibiting the replication of MERS-CoV efficiently in cells. For efficient delivery and expression, we cloned these effective amiRNAs into an adenoviral vector, termed Ad5, respectively to evaluate the immune protection in cell and rhesus monkey models. According to this study, we colud got a novel biological agent against MERS-CoV, and provide basic research data for MERS-CoV control.

2012年中东爆发了MERS-CoV疫情，逐渐蔓延到欧洲、非洲等地，形势不断恶化，已经引起了全世界极大关注。截至目前，全球已有184例确诊病例，死亡80人。面对日趋严峻的疫情，开发研制安全、有效的疫苗是十分必要的。RNAi由于可以特异性地抑制基因表达, 近年来越来越多的用于抗病毒治疗及预防的研究。在本研究中设计针对MERS-CoV的S基因、N基因的microRNA，首先在细胞水平上初步筛选出有效抑制MERS-CoV复制的microRNA。继而选用腺病毒作为将microRNA安全高效的运输载体，进一步研究重组腺病毒在细胞和恒河猴模型中对MERS-CoV的抑制效果，探讨其在病毒感染免疫保护中的作用。通过本研究的开展，以期获得一种新型抗MERS-CoV制剂，为研究MERS-CoV预防和治疗的新策略提供理论依据。

2012年爆发于中东的MERS-CoV疫情已经蔓延到欧洲、非洲、美洲等地，形势不断恶化，引起了人们的极大关注。面对严峻的疫情，开发研制安全、有效的疫苗和药物是十分必要的。本研究前期利用RNAi技术，设计针对MERS-CoV的S基因、N基因的microRNA，在细胞水平上初步筛选出有效抑制MERS-CoV S基因、N基因复制的microRNA，并以此构建腺病毒，扩增获得高滴度的病毒。将获得的重组腺病毒首先在MERS-CoV小鼠模型上进行了抗病毒研究，并未筛选出具有良好抑制效果的重组腺病毒，鉴于此并未在恒河猴上进行实验。.本研究建立了MERS-CoV小鼠感染模型，在病理变化上主要表现为急性肺炎，与临床报道的MERS-CoV感染的病人肺脏病理改变相似，表明该小鼠模型可以用于MERS-CoV致病机理及致病过程的研究。利用该模型评价了重组NTD疫苗，表明重组NTD蛋白在小鼠体内可诱导有效和持久的体液免疫和细胞免疫应答，并可部分保护小鼠免受MERS-CoV攻击，可作为MERS-CoV疫苗的候选。.本研究利用建立的MERS-CoV狨猴模型评价了麦考酚酸酯, 洛匹那韦/利托那韦和干扰素β1b的抗病毒效果。结果发现洛匹那韦/利托那韦治疗组和干扰素β1b治疗组取得了良好的治疗效果。两组均表现了较轻的临床症状，肺部产生中度病变，肺部病毒载量和36h死亡率均较未治疗组下降。本研究为MERS-CoV的临床用药提供了参考。.本研究还涉及一部分禽流感病毒流行病学进行研究，主要关注候鸟、家禽、留鸟之间的流感传播链，所获成果为禽流感的综合防控提供了理论依据。.总之，本研究在MERS-CoV小鼠模型建立、新型疫苗研发、药物评价进行研究，为MERS-CoV致病机制和综合防控奠定了基础。流感传播链的研究为禽流感的有效防控提供了参考。