Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate.
Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate.
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DOI：
10.1128/mbio.00650-13
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发表时间：
2013-09-10
期刊：
影响因子：
6.4
通讯作者：
Enjuanes L
中科院分区：
文献类型：
--
作者：
Almazán F;DeDiego ML;Sola I;Zuñiga S;Nieto-Torres JL;Marquez-Jurado S;Andrés G;Enjuanes L
中东呼吸综合征冠状病毒（MERS-CoV）是一种感染人类的新型冠状病毒，与急性肺炎、偶发性肾衰竭和高死亡率有关，被认为是对公共卫生的威胁。在细菌人工染色体中构建MERS-CoV基因组的全长感染性cDNA克隆，为研究病毒的分子生物学和开发减毒病毒作为疫苗候选物提供了反向遗传学系统。用cDNA克隆转染后，在Vero A66和Huh-7细胞中拯救了感染性病毒。缺失辅助基因3、4a、4 b和5的重组MERS-CoV（rMERS-CoV）成功地从缺失这些基因的cDNA克隆中拯救出来。突变病毒的生长动力学与野生型病毒相似，这表明辅助基因对于MERS-CoV在细胞培养物中的复制并不是必需的。相比之下，缺乏结构E蛋白的工程突变病毒（rMERS-CoV-ΔE）没有成功拯救，因为病毒感染性在早期传代时丧失。有趣的是，rMERS-CoV-ΔE基因组在cDNA克隆转染细胞后复制。感染性病毒被拯救并在表达E蛋白的细胞中繁殖，表明该病毒具有复制能力和繁殖缺陷。因此，rMERS-CoV-ΔE突变体病毒是潜在的预防MERS-CoV感染的安全且有希望的候选疫苗。自2012年夏季中东呼吸综合征冠状病毒在阿拉伯半岛出现以来，它已经传播到10个不同的国家，感染了大约94人，死亡率超过50%。本文介绍了第一个MERS冠状病毒的反向遗传学系统的开发，基于构建插入细菌人工染色体的感染性cDNA克隆。使用该系统，已经产生了rMERS-CoV缺失突变体的集合。有趣的是，其中一个E基因缺失的突变体是一种具有复制能力、繁殖缺陷的病毒，只能通过提供反式E蛋白在实验室中生长，而它只能在体内存活一个病毒感染周期。该病毒构成了候选疫苗，可能代表了诱导粘膜免疫的安全性和有效性之间的平衡，这是预防MERS-CoV感染所需的。自2012年夏季中东呼吸综合征冠状病毒在阿拉伯半岛出现以来，它已经传播到10个不同的国家，感染了大约94人，死亡率超过50%。本文介绍了第一个MERS冠状病毒的反向遗传学系统的开发，基于构建插入细菌人工染色体的感染性cDNA克隆。使用该系统，已经产生了rMERS-CoV缺失突变体的集合。有趣的是，其中一个E基因缺失的突变体是一种具有复制能力、繁殖缺陷的病毒，只能通过提供反式E蛋白在实验室中生长，而它只能在体内存活一个病毒感染周期。该病毒构成了候选疫苗，可能代表了诱导粘膜免疫的安全性和有效性之间的平衡，这是预防MERS-CoV感染所需的。
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection. Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.