Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
mBio. 2013 Sep 10;4(5):e00650-13. doi: 10.1128/mBio.00650-13.
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.
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.
中东呼吸综合征冠状病毒（MERS-CoV）是一种新兴的感染人类的冠状病毒，与急性肺炎、偶尔的肾衰竭和高死亡率有关，被认为是对公共健康的威胁。本文报道了一种全长传染性 cDNA 克隆的构建，该克隆插入细菌人工染色体，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一个反向遗传学系统。在转染 cDNA 克隆后，在 Vero A66 和 Huh-7 细胞中均可拯救出感染性病毒。从缺失这些基因的 cDNA 克隆中成功拯救出缺失辅助基因 3、4a、4b 和 5 的重组 MERS-CoV（rMERS-CoV）。突变病毒的生长动力学与野生型病毒相似，表明辅助基因不是 MERS-CoV 在细胞培养中复制所必需的。相比之下，缺失结构 E 蛋白的工程突变病毒（rMERS-CoV-ΔE）未能成功拯救，因为在早期传代时病毒感染力丧失。有趣的是，在将 cDNA 克隆转染到细胞中后，rMERS-CoV-ΔE 基因组仍能复制。在转染细胞中表达 E 蛋白的情况下，可拯救并增殖有感染性的病毒，表明该病毒具有复制能力但增殖缺陷。因此，rMERS-CoV-ΔE 突变病毒可能是一种安全且有前途的疫苗候选物，可预防 MERS-CoV 感染。
自 2012 年夏季中东 MERS-CoV 在阿拉伯半岛出现以来，它已传播到 10 个不同的国家，感染了约 94 人，死亡率高于 50%。本文描述了基于插入细菌人工染色体的传染性 cDNA 克隆构建的 MERS-CoV 第一个反向遗传学系统的开发。利用该系统，生成了一组 rMERS-CoV 缺失突变体。有趣的是，其中一个缺失 E 基因的突变体是一种复制能力完整但增殖缺陷的病毒，只能通过提供 E 蛋白在转染时才能在实验室中生长，而在体内它只能存活一个病毒感染周期。这种病毒构成了一种疫苗候选物，它可能在安全性和功效之间取得平衡，从而诱导黏膜免疫，这是预防 MERS-CoV 感染所必需的。
