Growth, detection, quantification, and inactivation of SARS-CoV-2

• Published in: Virology (New York, N.Y.) Vol. 548; pp. 39 - 48
• Main Authors: Case, James Brett, Bailey, Adam L., Kim, Arthur S., Chen, Rita E., Diamond, Michael S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2020; The Author(s). Published by Elsevier Inc
• Subjects: Animals; antigens; Antigens, Viral - analysis; Betacoronavirus - genetics; Betacoronavirus - growth & development; Betacoronavirus - immunology; Betacoronavirus - physiology; Cell Line; Chlorocebus aethiops; Containment of Biohazards; Coronavirus; COVID-19 infection; Culture Media; experimental design; Flow Cytometry; Focus-forming assay; immunity; pandemic; Plaque assay; quantitative polymerase chain reaction; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Viral - analysis; SARS-CoV-2; Severe acute respiratory syndrome coronavirus; Severe acute respiratory syndrome coronavirus 2; Titration; Vero Cells; Viral Plaque Assay; Virus Cultivation - methods; Virus Inactivation; Virus Replication; viruses; Flow cytometry; SARS-CoV-2; Coronavirus; Focus-forming assay; Virus inactivation; Titration; Plaque assay
• ISSN: 0042-6822; 1096-0341; 1096-0341
• DOI: 10.1016/j.virol.2020.05.015

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.