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      Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2

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          Abstract

          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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          Author and article information

          Contributors
          Journal
          Genes Dis
          Genes Dis
          Genes & Diseases
          Chongqing Medical University. Production and hosting by Elsevier B.V.
          2352-4820
          2352-3042
          17 July 2020
          17 July 2020
          Affiliations
          [1]Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China
          Author notes
          []Corresponding author. Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China. Tel.: +86 23-68486780; fax: +86 23-68486780.. wangkai@ 123456cqmu.edu.cn
          [∗∗ ]Corresponding author. Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China. Tel.: +86 23-68486780; fax: +86 23-68486780.. nitang@ 123456cqmu.edu.cn
          [1]

          These authors contributed equally to this work.

          Article
          S2352-3042(20)30089-1
          10.1016/j.gendis.2020.07.006
          7366953
          32837985
          0f4c276c-b843-419b-ab21-785abec016bd
          © 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.

          Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

          History
          : 26 May 2020
          : 8 July 2020
          : 13 July 2020
          Categories
          Article

          antiviral therapeutics,coronavirus,neutralizing antibodies,pseudovirus,sars-cov-2,spike protein

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