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

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...

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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
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Abstract	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.
AbstractList	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. 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.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.
Author	Case, James Brett Kim, Arthur S. Chen, Rita E. Diamond, Michael S. Bailey, Adam L.
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Keywords	Flow cytometry SARS-CoV-2 Coronavirus Focus-forming assay Virus inactivation Titration Plaque assay
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Snippet	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...
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SubjectTerms	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
Title	Growth, detection, quantification, and inactivation of SARS-CoV-2
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