Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark.
Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Antimicrob Agents Chemother. 2021 Jun 17;65(7):e0009721. doi: 10.1128/AAC.00097-21.
Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations , the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.
努力减轻 2019 年冠状病毒病（COVID-19）大流行包括筛选现有的抗病毒分子，可以将其重新用于治疗严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）感染。尽管 SARS-CoV-2 在非洲绿猴肾（Vero）细胞中高效复制和传播，但核苷（酸）类似物（NUC）等抗病毒药物由于代谢效率低下，通常在这些细胞中的活性降低。SARS-CoV-2 在培养的人细胞中存活能力较低。在这里，在人肝癌细胞克隆 Huh7.5 中连续传代 SARS-CoV-2 分离株（原始-SARS2）导致选择了一种变体（适应-SARS2），该变体在人肝（Huh7 和 Huh7.5）和肺癌（未修饰的 Calu-1 和 A549）细胞中具有显著提高的感染力。适应的病毒在刺突蛋白中具有突变，包括 9 个氨基酸缺失和 3 个氨基酸变化（E484D、P812R 和 Q954H）。E484D 也出现在 Vero E6 培养的病毒中，这些病毒在 A549 细胞中具有活力。原始和适应的病毒均易受清道夫受体 B 型 1（SR-B1）受体阻断，适应-SARS2 对 ACE2 的依赖性明显降低。两种变体均被 COVID-19 恢复期血浆类似地中和，但适应-SARS2 对外源 I 型干扰素的敏感性增加。瑞德西韦对原始和适应-SARS2 的抑制作用相似，证明了该系统用于筛选 NUC 的实用性。在所测试的 NUC 中，只有瑞德西韦、莫努匹韦和在一定程度上加拉迪韦在人细胞系中显示出抗病毒作用，而索非布韦、利巴韦林和法匹拉韦没有明显的活性。类似于刺突突变的出现，刺突蛋白在细胞培养中受到强烈的适应性选择压力。我们的结果表明，刺突突变的出现不太可能影响瑞德西韦的活性。
