The SARS-CoV-2 Spike is a virulence determinant and plays a major role on the attenuated phenotype of Omicron virus in a feline model of infection

To assess the role of the Omicron BA.1 Spike (S) protein in the pathogenesis of the severe acute respiratory coronavirus 2 (SARS-CoV-2), we generated recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 S gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 strain genome. The recombinant viruses were characterized in vitro and in vivo. Viral entry, cell-cell fusion, viral plaque size, and viral replication kinetics of the rWA1-Omi-S virus were markedly impaired when compared to the rWA1-D614G virus, demonstrating a lower fusogenicity and ability to spread cell-to-cell of rWA1-Omi-S. To assess the contribution of the Omicron BA.1 S protein to SARS-CoV-2 pathogenesis the pathogenicity of rWA1-D614G and rWA1-Omi-S viruses were compared using a feline model of infection. While the rWA1-D614G-inoculated cats became lethargic and showed increased body temperatures on days 2 and 3 post-infection (pi), rWA1-Omi-S-inoculated cats remained subclinical and gained weight throughout the 14-day experimental period. Animals inoculated with rWA1-D614G presented higher levels of infectious virus shedding in nasal secretions, when compared to rWA1-Omi-S-inoculated animals. In addition, tissue replication of the rWA1-Omi-S was markedly reduced compared to the rWA1-D614G, as evidenced by lower in situ viral RNA and lower viral load in tissues on days 3 and 5 pi. Histologic examination of the nasal turbinate and lungs revealed intense inflammatory infiltration in rWA1-D614G-inoculated animals, whereas rWA1-Omi-S-inoculated cats presented only mild to modest inflammation. Together, these results demonstrate that the S protein is a major virulence determinant for SARS-CoV-2 playing a major role for the attenuated phenotype of the Omicron virus. Author summaryThe SARS-CoV-2 Omicron sublineage BA.1 spread rapidly across the globe in late 2021/early 2022. Experimental studies have shown an overall lower pathogenicity of Omicron BA.1 when compared to the ancestral SARS-CoV-2 lineage B.1 (D614G). Recently, we have demonstrated that the Omicron BA.1.1 variant presents lower pathogenicity when compared to D614G (B.1) lineage in a feline model of SARS-CoV-2 infection. There are over 50 mutations in the Omicron genome, of which more than two thirds are present in the S gene. To assess the role of the Omicron BA.1 S on virus pathogenesis, recombinant viruses harboring the S D614G mutation (rWA1-D614G) and the Omicron BA.1 Spike gene (rWA1-Omi-S) in the backbone of the ancestral SARS-CoV-2 WA1 were characterized in vitro and in vivo. While the Omicron BA.1 S gene results in early entry into cells, the rWA1-Omi-S presents impaired cell-cell spread and fusogenic activity. Inoculation of cats with the recombinant viruses revealed an attenuated phenotype of rWA1-Omi-S, demonstrating a critical role for S protein on the pathogenicity of SARS-CoV-2 and indicating that the Omi-S is a major determinant of the attenuated disease phenotype of Omicron strains.