Korea Zoonosis Research Institute and Genetic Engineering Research Institute, Chonbuk National University, Jeonju 561-756, Republic of Korea.
J Microbiol Biotechnol. 2019 Jun 28;29(6):999-1007. doi: 10.4014/jmb.0905.05061.
Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virusspecific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patientisolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.
中东呼吸综合征冠状病毒（MERS-CoV）于 2012 年在沙特阿拉伯首次被发现，此后已有 20 多个国家报告了相关感染病例。迄今为止，全球共报告约 10000 例人类感染病例，死亡率高达 40%。大多数病例发生在沙特阿拉伯，除了 2015 年韩国的一次疫情外，其他国家都有零星爆发。韩国 MERS-CoV 株从第二名韩国患者中分离出来，其基因组已被完全测序并保存。为了开发针对韩国分离株的病毒特异性保护和治疗药物，并研究病毒-宿主相互作用的分子决定因素，生成其全长 cDNA 至关重要。在这里，我们报告说，通过常规克隆技术和高效的 Gibson 组装反应相结合，从一名韩国患者中分离出的 MERS-CoV 株生成了两个全长 cDNA。通过限制性分析验证了全长 cDNA 的完整性，并通过 Sanger 法验证了其序列。所得 cDNA 可在体外有效转录，且 T7 启动子驱动的表达非常稳健。该反向遗传系统将补充已发表的 MERS-CoV cDNA 列表，并有助于开发针对韩国分离株的抗病毒措施。
