Abstract: Objective To investigate the molecular mechanisms underlying the deletion of the Furin Cleavage Site (FCS) during in vitro culturing of SARS-CoV-2 and to analyze the biological significance of this phenomenon for the virus's origin and evolution. Methods The prototype strain and various mutant strains of SARS-CoV-2 were serially passaged in Vero E6 and 293T-hACE2 cell lines. High-throughput sequencing of multiplex PCR amplicons was employed to dynamically monitor the variation characteristics of the FCS region and its flanking sequences. The association between FCS deletion, the viral entry pathway, and specific genomic loci was analyzed by pharmacologically inhibiting the Transmembrane Serine Protease 2 (TMPRSS2) pathway. Phylogenetic analysis was conducted in conjunction with sequences from global public databases. Results The in vitro serial passage experiments demonstrated that, with the exception of B.1、Alpha, Omicron, and their sublineages, all other tested SARS-CoV-2 evolutionary branches underwent rapid deletion of the FCS sequence. Although direct FCS deletion was not detected after six passages (P6) of plaque-purified B.1 virus in both cell lines, a mixed T/C quasispecies emerged at position 23525 of the spike (S) protein gene, with its mutation frequency increasing with successive passages. Following the blockade of the TMPRSS2-dependent cell surface entry pathway, mutant strains with FCS deletions rapidly appeared and were enriched, becoming the dominant population (reaching an abundance of 57.9%) by P6. Sequence analysis confirmed a strict co-occurrence of the FCS deletion with synergistic T>C mutations at upstream positions 23422 and 23525, indicating that a cytosine (C) genotype at this dual-locus is possibly a prerequisite molecular event for the FCS deletion. Database analysis further revealed that position 23422 is highly conserved in human populations, whereas position 23525 exhibits lineage-specificity: early epidemic strains (e.g., Delta) predominantly carried 23525C and were prone to FCS deletion, while later strains (e.g., Omicron) predominantly featured 23525T with a relatively stable FCS, observations that are highly consistent with the in vitro experimental phenotypes. Conclusion The deletion of the FCS during in vitro replication of SARS-CoV-2 exhibits marked sequence specificity. The concurrent presence of a cytosine (C) genotype at the upstream 23422 and 23525 loci is possibly a critical molecular precondition that induces the deletion in this region. This study elucidates the molecular determinants of FCS stability, providing a significant basis for clarifying the adaptive mechanisms of SARS-CoV-2 in cross-species transmission and its molecular evolutionary patterns.

Key words: SARS-CoV-2, Furin cleavage site (FCS), Deletion mechanism, High-throughput sequencing