Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol.
Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol.
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DOI：
10.3389/fcvm.2020.618651
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发表时间：
2020
影响因子：
3.6
通讯作者：
Shehadeh LA
中科院分区：
文献类型：
--
作者：
Condor Capcha JM;Lambert G;Dykxhoorn DM;Salerno AG;Hare JM;Whitt MA;Pahwa S;Jayaweera DT;Shehadeh LA
由SARS-CoV-2冠状病毒引起的COVID-19大流行需要可靠的检测方法来研究病毒进入机制，但对该机制的了解仍然很少。这一知识对于通过筛选中和抗体和其他可以阻断感染的药物来控制SARS-CoV-2感染的治疗方法的发展非常重要。这对于处于COVID-19相关严重结局高风险的患者尤为重要。对于没有两种最常用的假型系统（即逆转录病毒/慢病毒和缺乏VSV包膜糖蛋白（VSVΔG）的水泡性口炎病毒（VSV））经验的非病毒学实验室来说，假型病毒颗粒的生产似乎是一项艰巨的任务。通过整合最新的知识，我们已经开发了一个详细的，易于遵循的新协议，用于生产SARS-CoV-2刺突轴承假病毒使用VSV-ΔG系统。我们描述了感染检测，其使用GFP荧光作为在24孔实时成像系统中的感染的量度。我们目前的结果，我们优化的系统，以提高病毒感染水平，通过过表达的人ACE 2受体和过表达的至少一个的两种蛋白酶-TMPRSS 2或弗林蛋白酶，以及补充泊洛沙姆407（P407）和前列腺素E2（PGE 2）作为佐剂。我们表明，该系统有效地工作在三个无关的，临床相关的细胞系：人293 T（肾上皮）细胞，人Calu-3（肺上皮）细胞，和非人灵长类动物（非洲绿色猴）细胞系，Vero-E6（肾上皮）细胞。此外，我们已经使用该系统来显示人诱导多能干细胞衍生的心肌细胞（iPS-CM）的感染。该系统是有效的（病毒生成，滴定和感染测定可以在1周内进行），定量，廉价，并且易于扩展应用于药物开发和治疗筛选方法。
The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches.
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DOI：
10.1007/978-1-59745-181-9_23
发表时间：
2008
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Fukushi S;Watanabe R;Taguchi F
通讯作者：
Taguchi F
影响因子：
7.6
作者：
Liu R;Wang J;Shao Y;Wang X;Zhang H;Shuai L;Ge J;Wen Z;Bu Z
通讯作者：
Bu Z
影响因子：
28.3
作者：
Gorbalenya, Alexander E.;Baker, Susan C.;Ziebuhr, John
通讯作者：
Ziebuhr, John
影响因子：
8
作者：
McAndrews, Kathleen M.;Dowlatshahi, Dara P.;Kalluri, Raghu
通讯作者：
Kalluri, Raghu
影响因子：
5.4
作者：
Johnson MC;Lyddon TD;Suarez R;Salcedo B;LePique M;Graham M;Ricana C;Robinson C;Ritter DG
通讯作者：
Ritter DG