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.

Keywords: Calu-3; SARS-CoV-2; polymorphisms; stock preparation.

Figure 1

Schematic of the passage history for the virus stocks characterized in this study. Created with BioRender.com.

Figure 2

Hamsters are susceptible to pathogenic infection with BQ-RSA-p4 grown on Calu-3 cells. (A) The mean and SEM of the body weight for all four hamsters infected with BQ-RSA-p4 over the 7 days post challenge are shown. The dotted line represents zero change in body weight. Total (B) and subgenomic (C) viral copies from oral swabs were measured during the course of infection for all four hamsters listed. On the day of necropsy, the total and subgenomic viral copies were measured in the bronchoalveolar lavage fluid (D) and lung tissue (E) for all four hamsters. Each dot represents a different animal, and the median is shown with the line. Methods from Alleva et al. [24] were used for virus quantification.