SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.
SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.
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DOI：
10.1099/jgv.0.001481
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发表时间：
2020-11
期刊：
影响因子：
--
通讯作者：
Duprex WP
中科院分区：
文献类型：
--
作者：
Klimstra WB;Tilston-Lunel NL;Nambulli S;Boslett J;McMillen CM;Gilliland T;Dunn MD;Sun C;Wheeler SE;Wells A;Hartman AL;McElroy AK;Reed DS;Rennick LJ;Duprex WP
严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是 2019 年冠状病毒病 (COVID-19) 的病原体，于 2019 年底出现，到 2020 年 6 月中旬，该病毒已传播到至少 215 个国家，造成超过 8 万例确诊感染和超过 45 万例死亡，并使全球医疗保健系统不堪重负。与 2002 年出现并引起类似疾病的严重急性呼吸综合征冠状病毒 (SARS-CoV) 一样，SARS-CoV-2 是一种 β 冠状病毒。这两种病毒都使用人类血管紧张素转换酶2（hACE2）作为受体进入细胞。然而，SARS-CoV-2 刺突 (S) 糖蛋白有一个新的插入，可产生假定的弗林蛋白酶裂解信号，这被认为可以扩大宿主范围。 SARS-CoV-2 的两种低传代 (P) 毒株（Wash1:P4 和 Munich:P1）在 Vero E6 细胞中培养两次并进行病毒学表征。 Sanger 和 MinION 测序表明 Wash1:P6 的弗林蛋白酶裂解信号存在显着缺失，而慕尼黑：P3 菌株中存在微小变异。即使存在完整的弗林蛋白酶裂解信号，SARS-CoV-2 感染的 Vero E6 细胞裂解物中 S 糖蛋白的裂解效率也很低。间接免疫荧光证明S糖蛋白到达细胞表面。由于 S 蛋白是中和抗体开发的主要抗原靶标，因此我们研究了从 COVID-19 人类患者获得的系列血清样本中中和抗体滴度的变化。无论是否存在完整或缺失的弗林蛋白酶裂解信号，这些结果都是可比的。这些研究表明，在进行体外或体内发病机制研究之前，需要仔细表征病毒库。
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.
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