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Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike

Wisconsin National Primate Center, UW-Madison, Madison, WI 53711, USA
BIOQUAL, Inc., Rockville, MD 20852, USA
American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, USA
The Biodefense and Emerging Infections Research Resources Repository (BEI Resources), 10801 University Boulevard, Manassas, VA 20110, USA
Office of Biodefense, Research Resources and Translational Research, Division of Microbiology and Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA
Department of Pathology and Laboratory Medicine, UW-Madison, Madison, WI 53705, USA
Author to whom correspondence should be addressed.
These authors contributed equally to the manuscript.
Academic Editor: Shibo Jiang
Viruses 2021, 13(12), 2434;
Received: 16 November 2021 / Revised: 2 December 2021 / Accepted: 2 December 2021 / Published: 4 December 2021
(This article belongs to the Topic Infectious Diseases)
SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation. View Full-Text
Keywords: SARS-CoV-2; polymorphisms; Calu-3; stock preparation SARS-CoV-2; polymorphisms; Calu-3; stock preparation
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MDPI and ACS Style

Baczenas, J.J.; Andersen, H.; Rashid, S.; Yarmosh, D.; Puthuveetil, N.; Parker, M.; Bradford, R.; Florence, C.; Stemple, K.J.; Lewis, M.G.; O’Connor, S.L. Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike. Viruses 2021, 13, 2434.

AMA Style

Baczenas JJ, Andersen H, Rashid S, Yarmosh D, Puthuveetil N, Parker M, Bradford R, Florence C, Stemple KJ, Lewis MG, O’Connor SL. Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike. Viruses. 2021; 13(12):2434.

Chicago/Turabian Style

Baczenas, John J., Hanne Andersen, Sujatha Rashid, David Yarmosh, Nikhita Puthuveetil, Michael Parker, Rebecca Bradford, Clint Florence, Kimberly J. Stemple, Mark G. Lewis, and Shelby L. O’Connor. 2021. "Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike" Viruses 13, no. 12: 2434.

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