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

Abstract

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.

A Deletion Encompassing the Furin Cleavage Site in the Spike Encoding Gene Does Not Alter SARS-CoV-2 Replication in Lung Tissues of Mink and Neutralization by Convalescent Human Serum Samples

“…Importantly, this report also highlights the importance of sequencing viral stocks before in vivo, in vitro and ex vivo studies as deletions may occur thus altering results and their interpretation. In this perspective, the use of human lung adenocarcinoma cells expressing TMPRSS2 (Calu-3) is strongly recommended to avoid the onset of undesired mutations in the spike protein encoding gene including the CS [ 25 , 26 , 27 ].…”

Choosing a cellular model to study SARS-CoV-2

“…In addition to these two disadvantages of using Calu-3 Cells, a third can also be mentioned. Calu-3 cells grow more slowly than Vero cells (Baczenas et al, 2021), but this does not mean they are inferior to Vero cells. Instead, this is because, unlike in Vero cells, the furin cleavage site appears to be preserved in SARS-CoV-2 produced in Calu-3 cells, rather than promoting the attenuation typically observed during passages in Vero cells (Baczenas et al, 2021;Lamers et al, 2021;Mykytyn et al, 2021a).…”

“…This isolated virus can also enable the production of antigens or can be employed in antiviral susceptibility screening tests (Leland and Ginocchio, 2007). However, this posterior assay requires a well-characterised viral stock produced in cell culture, ideally with minimal passaging to reduce the rise of genetic subpopulations and heterogenicity of phenotypes of the virus stock (Baczenas et al, 2021). Therefore, this virus stock will serve as a seed stock which will be propagated to make more extensive stocks.…”

“…This type of feature makes Calu-3 cells interesting for SARS-CoV-2 studies, particularly if this stock is being produced for infections in animal models, as Calu-3-derived virus stocks remained pathogenic in hamsters, and the Calu-3-specific variants were maintained (Baczenas et al, 2021). In addition, these cells are also widely used in drug screening studies against SARS-CoV-2 (Dittmar et al, 2021;Tao et al, 2021;Coimbra et al, 2022).…”

The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense

“…For single cell RNAseq of the mixed organoids, SARS-CoV-2 isolate WA1 (USA-WA1/2020, A) was acquired from BEI and propagated on VeroE6 cells resulting in mutation of the furin cleavage site ( 144, 145 ).…”