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Title: [Molecular epidemiological study of human coronavirus OC43 in Shanghai from 2009-2016].
Author: Yang YJ, Hu YW.
Journal: Zhonghua Yu Fang Yi Xue Za Zhi; 2018 Jan 06; 52(1):55-61. PubMed ID: 29334709.
Abstract:
Objective: To understand the epidemiological characteristics of Human coronavirus (HCoV), the patterns of emergence and circulation, and the genotype distribution of human coronavirus OC43 (HCoV-OC43) from November, 2009 to April, 2016 in Shanghai. Methods: A total of 6 059 respiratory specimens, including pharyngeal swab, sputum, nasopharyngeal aspirates and alveolar lavage fluid, as well as relative clinical data were collected from patients with acute respiratory infections from seven sentinel hospitals during November, 2009 to April, 2016 in Shanghai. Respiratory specimens were tested by RT-PCR with HCoV-conserved primers and subsequently genotyped by DNA sequencing. Using specific primers to amplify and sequence full-length Spike (S), RNA-dependent RNA polymerase (RDRP) and nucleocapsid (N) gene from HCoV-OC43 positive samples. Further genotype and phylogenetic analysis of HCoV-OC43 were performed by conducting phylogenetic trees. Results: Among 6 059 patients, the total frequency of HCoV was 63 (1.04%), in which HCoV-OC43 was the most frequently detected species with 34 positive samples, followed by human coronavirus 229E (HCoV-229E) and human coronavirus HKU1 (HCoV-HKU1) with 18 and 10 positive sample respectively. However, other HCoV like human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV), were not been detected, which illustrated that HCoV-OC43 was the dominant subtype. The full-length of S, RDRP and N gene were obtained from 29 HCoV-OC43 positive samples. According to the sequence-analysis, 27 of which was genotype D, 2 of which was genotype B and others genotype, including genotype E, F and G, were not detected. The result indicated that the genotype D may be the dominant genotype. Further analysis of S protein that help HCoV-OC43 to entry host cell and stimulate the host immune system to produce neutralizing antibody found that two important functional domains in S protein, N-terminal domain (NTD) and receptor-binding domain (RBD) contained more amino acid substitution and positive selection sites, accompanied with amino acid insertion/deletion. 13 positive selection sites were all located in the NTD or RBD, 10 of which were located in the NTD and 3 in the RBD. Conclusion: Human coronavirus OC43 was the major circulation human coronaviurs in Shanghai from 2009 to 2016, in which genotype D was the dominant genotype. NTD and RBD regions of the S protein were hypervariable region during HCoV-OC43 evolution, and had amino acid substitutions as well as amino acid insertion/deletion. 目的： 分析上海2009年11月至2016年4月人冠状病毒（HCoV）流行特征，以及HCoV-OC43基因型分布和变异变迁规律。 方法： 收集2009年11月至2016年4月期间上海7家哨点医院感染科急性呼吸道感染患者的临床资料和呼吸道样本，包括咽拭子、痰、鼻咽抽吸物和肺泡灌洗液，共6 059例。采用HCoV通用引物对患者样本进行检测并测序分型。HCoV-OC43阳性样本进一步采用特异性引物对刺突蛋白、依赖RNA的RNA聚合酶（RDRP）和核衣壳蛋白全基因进行扩增和测序，并通过全基因序列构建进化树对HCoV-OC43进行基因分型和进化分析。 结果： 共检出HCoV 63株（1.04%），其中HCoV-OC43检出数最多，为34株，其后依次是HCoV-229E和HCoV-HKU1，检出数分别为18和10株，而HCoV-NL63、重症急性呼吸综合征冠状病毒（SARS-CoV）和中东呼吸综合征冠状病毒（MERS-CoV）均未检出。29例HCoV-OC43阳性样本获得刺突蛋白、RDRP和核衣壳蛋白全基因序列，根据进化树分析显示，其中27例为D型，2例为B型，而其他基因型E、F、G均未检出，其中D型为主导基因型。进一步分析与HCoV-OC43进入宿主和中和抗体产生相关的刺突蛋白发现，刺突蛋白重要的功能结构域——N端结构域（NTD）和受体结合结构域（RBD）含有较多氨基酸变异和阳性选择位点，并伴有氨基酸插入/缺失。13个阳性选择位点均位于NTD或RBD，其中10个位于NTD，3个位于RBD。 结论： 2009—2016年，上海流行的HCoV主要为HCoV-OC43，其中D型为优势基因型。刺突蛋白的NTD区和RBD区是HCoV-OC43进化过程中的高变区域，伴有氨基酸替换、氨基酸插入/缺失。.

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