A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells

Abstract

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

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

“…At necropsy, we detected approximately 2 × 10 9 copies of virus per gram of lung tissues, consistent with other studies of the SARS-CoV-2 infection of hamsters (Figure 2) [15,[25][26][27]. Additionally, infected hamsters lost 15% of their body weight during the study, as expected for a pathogenic SARS-CoV-2 that has an intact furin cleavage site [14,15]. At necropsy, lungs were collected, and RNA was isolated for sequencing.…”

“…These hamsters lost ~15% of their body weight, further suggesting that the Calu-3-associated mutations did not affect virus pathogenicity in hamsters. This stands in stark contrast to the limited weight loss observed in hamsters infected with stocks lacking a furin cleavage site [14,15]. The variants detected in the Calu-3-derived stocks were also either maintained or expanded in the hamsters.…”

“…Stocks Propagated by BIOQUAL SARS-CoV-2 inocula were sequenced using a modified approach originally developed by ARTIC Network (https://artic.network, accessed on 12 February 2021) [14]. Briefly, complementary DNA (cDNA) was synthesized with SuperScript IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), random hexamers, and dNTPs.…”

“…However, SARS-CoV-2 can also enter Vero cells by endocytosis, rendering the need to use the ACE2 receptor irrelevant [9,10]. Several groups have found that the propagation of SARS-CoV-2 on Veroderived cells lines leads to the rapid accumulation and loss of a functional furin cleavage site within a few passages [11][12][13][14]. As a result, the propagation of SARS-CoV-2 on Vero cells gave rise to challenge stocks with defects in the furin cleavage site, making them non-pathogenic in animals [15].…”

Standardised neutralising antibody assays are needed for evaluating COVID-19 vaccines

“…The gold standard method for measuring vaccine-induced neutralizing antibodies is virus neutralisation test and plaque reduction neutralisation test using live viruses. However, virus strains isolated from different laboratories are geographically restricted and often limited in number, and may undergo laboratory variation during culture [6] . Although pseudovirus-based neutralisation assays have a high practical value as an alternative method, the vectors for constructing pesudovirus and testing procedures are different in different laboratories.…”