Alotaibi Badriyah, El-Masry Thanaa A, Seadawy Mohamed G, El-Harty Bassem E, Saleh Asmaa, Gad Ahmed F, El-Hosseny Mostafa F, Mahran Yasmen F, El-Bouseary Maisra M
Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourahbint Abdulrahman University, Riyadh, Saudi Arabia.
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
Infect Genet Evol. 2022 Jun;100:105278. doi: 10.1016/j.meegid.2022.105278. Epub 2022 Mar 31.
The recently emerging coronavirus, severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Since its discovery in the city of Wahan, China, SARS-CoV-2 has spread rapidly to invade all countries. In addition to its rapid transmission rate, it is characterized by high genetic mutation rates. The aim of this study is to provide an effective method for the isolation and propagation of SARS-CoV-2 in cell lines without any induction of genetic variations. In this study, we isolated SARS-CoV-2 from oro-nasopharyngeal swabs collected from Egyptian patients who were clinically diagnosed with COVID-19. Molecular identification of SARS-CoV-2 was performed by Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). The isolated virus was propagated on Vero E6 cells without applying serial viral passages to avoid any variation of the viral genome. The replication and propagation were confirmed by the results of both RT-qPCR and the cytopathic effect (CPE). Moreover, SARS-CoV-2 was completely inactivated chemically using beta-propiolactone (βPL). Whole genome sequencing (WGS) of the propagated virus was performed in order to investigate mutational patterns. The genome sequences recovered in 2020 (n = 18) were similar to the reference strain, Wuhan-Hu-1, and were clustered as clade 20A. However, the genomic sequences recovered in 2021 (n = 2) were clustered as clade 21J. These two sequences are considered the first Delta (B.1.617.2) variants detected in Egypt. This study provides a reference for researchers in Egypt to isolate and propagate SARS-CoV-2 easily and efficiently. Furthermore, the prevalence of the SARS-CoV-2 delta variant in Egypt necessitates continuous monitoring of the efficacy of the applied treatment protocol and the effectiveness of current vaccines against such variants of concern (VOC).
最近出现的冠状病毒，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是2019冠状病毒病（COVID-19）大流行的病原体。自在中国武汉市被发现以来，SARS-CoV-2迅速传播，侵袭了所有国家。除了其快速传播率外，它还具有高基因突变率的特点。本研究的目的是提供一种在细胞系中分离和繁殖SARS-CoV-2的有效方法，而不引发任何基因变异。在本研究中，我们从临床诊断为COVID-19的埃及患者采集的口咽拭子中分离出SARS-CoV-2。通过实时定量逆转录聚合酶链反应（RT-qPCR）对SARS-CoV-2进行分子鉴定。分离出的病毒在Vero E6细胞上繁殖，不进行连续病毒传代，以避免病毒基因组发生任何变异。通过RT-qPCR结果和细胞病变效应（CPE）证实了病毒的复制和繁殖。此外，使用β-丙内酯（βPL）对SARS-CoV-2进行化学完全灭活。对繁殖的病毒进行全基因组测序（WGS），以研究突变模式。2020年获得的基因组序列（n = 18）与参考毒株武汉-Hu-1相似，聚类为20A分支。然而，2021年获得的基因组序列（n = 2）聚类为21J分支。这两个序列被认为是在埃及检测到的首批德尔塔（B.1.617.2）变体。本研究为埃及的研究人员轻松、高效地分离和繁殖SARS-CoV-2提供了参考。此外，SARS-CoV-2德尔塔变体在埃及的流行使得有必要持续监测所应用治疗方案的疗效以及当前疫苗针对此类关注变体（VOC）的有效性。
