Kossyvakis Athanasios, Tao Ying, Lu Xiaoyan, Pogka Vasiliki, Tsiodras Sotirios, Emmanouil Mary, Mentis Andreas F, Tong Suxiang, Erdman Dean D, Antoniadis Antonios
National Influenza Reference Laboratory of Southern Greece, Hellenic Pasteur Institute, Athens, Greece.
Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS One. 2015 Apr 28;10(4):e0125809. doi: 10.1371/journal.pone.0125809. eCollection 2015.
Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient's history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.
对疑似中东呼吸综合征冠状病毒（MERS-CoV）感染的人员进行快速可靠的实验室诊断，对于及时实施感染控制措施和疾病管理至关重要。此外，监测病毒的分子变化有助于阐明传播链，并识别可能影响病毒传播效率的突变。我们最近对希腊一例输入性MERS-CoV病例进行的实验室调查就说明了这一点。在患者住院的第1天和第2天采集了两份口咽拭子标本，并使用两种针对MERS-CoV基因组UpE和Orf-1a区域的实时逆转录聚合酶链反应（rRT-PCR）检测方法、RNA依赖的RNA聚合酶和核衣壳基因的逆转录聚合酶链反应（RT-PCR）及部分测序进行检测。还采集了血清标本并进行了血清学检测。第一份拭子样本的结果不确定，而第二份拭子通过rRT-PCR检测MERS-CoV RNA呈强阳性，并经RT-PCR和部分基因测序确认为阳性。血清学检测阳性结果进一步证实了MERS-CoV感染。随后从阳性拭子样本中获得了全长核衣壳和刺突基因编码序列。系统发育分析表明，该病毒与2014年4月在沙特阿拉伯吉达和麦加获得的近期人源MERS-CoV毒株以及沙特阿拉伯和卡塔尔的单峰骆驼密切相关。这些发现与患者的病史相符。我们还在刺突受体结合域中鉴定出一个独特的氨基酸替代，这可能对受体结合效率有影响。我们最初rRT-PCR结果不确定突出了从疑似MERS-CoV病例中采集多个标本尤其是下呼吸道标本的重要性。
