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[资源共享] SARS-CoV-2假病毒包装方法

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发表于 2020-8-24 16:12:35 | 显示全部楼层 |阅读模式
新冠假病毒包装方法:
1. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.
Emerging Microbes & Infections
https://doi.org/10.1080/22221751.2020.1743767  
  Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.




2. Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2.
Genes & Diseases
DOI: 10.1016/j.gendis.2020.07.006
https://doi.org/10.1016/j.gendis.2020.07.006

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of the coronavirus disease 2019 (COVID-19) pandemic. To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2, we constructed a codon-optimized, full-length C-terminal mutant spike (S) gene of SARS-CoV-2. We generated a luciferase (Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone. The key parameters for this pseudovirus-based assay, including the S mutants and virus incubation time, were optimized. This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2 (ACE2)-expressing 293T cells. Cathepsin (Cat)B/L inhibitor E−64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells. Furthermore, the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1. Thus, we developed a pseudovirus-based assay for SARS-CoV-2, which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.


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 楼主| 发表于 2020-8-26 08:40:53 | 显示全部楼层
3. Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro

https://www.sciencedirect.com/sc ... i/S2590053620300884

33

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526

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 楼主| 发表于 2020-9-14 08:49:30 | 显示全部楼层
4.Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells
https://www.tandfonline.com/doi/ ... 221751.2020.1815589
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