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[从土耳其安塔利亚获得的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)分离株的基因组特征分析]

[Genomic Characterization of SARS-CoV-2 Isolates Obtained from Antalya, Türkiye].

作者信息

Erman Daloğlu Aylin, Lizarazo Forero Erley, Sepin Özen Nevgün, Çekin Yeşim, Niesters Hubert G M

机构信息

University of Health Sciences, Antalya Training and Research Hospital, Medical Microbiology Clinic, Antalya, Türkiye.

The University of Groningen, University Medical Center Groningen, Department of Medical Microbiology and Infection Prevention, Division of Clinical Virology, Groningen, Netherlands.

出版信息

Mikrobiyol Bul. 2024 Oct;58(4):433-447. doi: 10.5578/mb.20249643.

Abstract

As the number of coronavirus diseases-2019 (COVID-19) cases have decreased and measures have started to be implemented at an individual level rather than in the form of social restrictions, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) still maintains its importance and has already taken its place in the spectrum of agents investigated in multiplex molecular test panels for respiratory tract infections in routine diagnostic use. In this study, we aimed to present mutation analysis and clade distribution of whole genome sequences from randomly selected samples that tested positive with SARS-CoV-2 specific real-time reverse transcription polymerase chain reaction (rRT-PCR) test at different periods of the pandemic in our laboratory with a commercial easy-to-use kit designed for next-generation sequencing systems. A total of 84 nasopharyngeal/oropharyngeal swab samples of COVID-19 suspected patients which were sent for routine diagnosis to the medical microbiology laboratory and detected as SARSCoV-2 RNA positive with rRT-PCR were randomly selected from different periods for sequence analysis. Library preparation for sequencing was performed with the commerical EasySeq SARS-CoV-2 RC PCR kit (Nimagen, the Netherlands). The data generated from the Illumina MiSeq system (Illumina Inc, San Diego, CA, USA) were analysed using CLC Genomics Workbench (CLC, Qiagen, Hilden, Germany). Nextstrain clades detected in order of frequency were 21J (Delta) (25%, n= 21), 21L (Omicron) (23.8%, n= 20), 20B (19%, n= 16), 20A (15.5%, n= 13), 21K (Omicron) (11.9%, n= 10), 19A (3.6%, n= 3), and 22B (Omicron) (1.2%, n= 1). Excluding one patient sample which was identified as 22B (Omicron), a total of 2829 common distinct mutations (2076 missense, 551 synonymous, 192 deletions and 10 insertions) were detected. 100 mutations were observed in the non-coding 5' untranslated region (UTR). The majority of the mutations were located in the Spike gene region (1120 mutations), followed by the ORF1a (624 mutations), nucleocapside (315 mutations) and ORF1b (263 mutations) gene regions. Sampling times of the patients were March 2020 (n= 1), April 2020 (n= 11), May 2020 (n= 1), June 2020 (n= 2), July 2020 (n= 3), August 2020 (n= 1), September 2020 (n= 5), November 2020 (n= 2), December 2020 (n= 6), December 2021 (n= 19), January 2022 (n= 11), March 2022 (n= 16), April 2022 (n= 3), and June 2022 (n= 3). As a result, in this study, SARS-CoV-2 variants and mutations in the Mediterranean Region of Türkiye, Antalya province were analyzed in detail. To the best of our knowledge, no SARS-CoV-2 genome analysis study from the pandemic period has been reported in our province. When the sequences from our study were uploaded to the GISAID Instant Audacity system and the related genomes obtained from different countries in the EpiCoV database metadata were examined, the top two countries in terms of similarity and which could be associated with the main entry route of the virus into Türkiye were Germany and the United Kingdom. In today's world, where it is discussed what can be done to be prepared for possible new pandemics based on the COVID-19 pandemic, the importance of being proactive in molecular surveillance studies is indisputable. Developing countries should be supported and encouraged to research new variants and share data in addition to known variants in pandemic control. At this point, we believe that past pandemic data reported from different geographical regions will also be valuable in terms of predicting the circulation network and taking precautions in a possible new pandemic.

摘要

随着2019冠状病毒病(COVID-19)病例数减少,且防控措施已开始在个人层面而非以社会限制的形式实施,但严重急性呼吸综合征冠状病毒2(SARS-CoV-2)仍然具有重要性,并且已在常规诊断中用于呼吸道感染的多重分子检测面板所研究的病原体谱中占据一席之地。在本研究中,我们旨在展示使用专为下一代测序系统设计的商用易用试剂盒,对我们实验室在疫情不同时期经SARS-CoV-2特异性实时逆转录聚合酶链反应(rRT-PCR)检测呈阳性的随机选择样本的全基因组序列进行突变分析和进化枝分布。从医学微生物实验室送去进行常规诊断并经rRT-PCR检测为SARS-CoV-2 RNA阳性的84例COVID-19疑似患者的鼻咽/口咽拭子样本中,从不同时期随机选择进行序列分析。使用商用EasySeq SARS-CoV-2 RC PCR试剂盒(荷兰Nimagen公司)进行测序文库制备。使用CLC Genomics Workbench(德国希尔德市Qiagen公司的CLC)分析Illumina MiSeq系统(美国加利福尼亚州圣地亚哥市Illumina公司)产生的数据。按频率顺序检测到的Nextstrain进化枝为21J(德尔塔)(25%,n = 21)、21L(奥密克戎)(23.8%,n = 20)、20B(19%,n = 16)、20A(15.5%,n = 13)、21K(奥密克戎)(11.9%,n = 10)、19A(3.6%,n = 3)和22B(奥密克戎)(1.2%,n = 1)。排除1例鉴定为22B(奥密克戎)的患者样本后,共检测到2829个常见的不同突变(2076个错义突变、551个同义突变、192个缺失和10个插入)。在非编码5'非翻译区(UTR)观察到100个突变。大多数突变位于刺突基因区域(1120个突变),其次是ORFla(624个突变)、核衣壳(315个突变)和ORF1b(263个突变)基因区域。患者的采样时间为2020年3月(n = 1)、2020年4月(n = 11)、2020年5月(n = 1)、2020年6月(n = 2)、2020年7月(n = 3)、2020年8月(n = 1)、2020年9月(n = 5)、2020年11月(n = 2)、2020年12月(n = 6)、2021年12月(n = 19)、2022年1月(n = 11)、2022年3月(n = 16)、2022年4月(n = 3)和2022年6月(n = 3)。结果,在本研究中,对土耳其安塔利亚省地中海地区的SARS-CoV-2变体和突变进行了详细分析。据我们所知,我省尚未有关于疫情期间SARS-CoV-2基因组分析的研究报道。当将我们研究中的序列上传到GISAID即时Audacity系统,并检查EpiCoV数据库元数据中从不同国家获得的相关基因组时,在相似性方面排名前两位且可能与病毒进入土耳其的主要传播途径相关的国家是德国和英国。在当今这个基于COVID-19疫情讨论如何为可能的新疫情做准备的世界中,在分子监测研究中积极主动的重要性是无可争议的。除了在疫情防控中研究已知变体之外,应该支持和鼓励发展中国家研究新变体并共享数据。在这一点上,我们相信不同地理区域报告的过去疫情数据对于预测传播网络以及在可能的新疫情中采取预防措施也将是有价值。

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