SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells

Abstract

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

Keywords: Delta variant; Omicron variant; SARS-CoV-2; TMPRSS2; viral replication.

Figure 1.

Viral replication in VeroE6/TMPRSS2, VeroE6 and Calu3 cells. (A) Viral replication kinetics in VeroE6/TMPRSS2 cells. (B) Viral replication kinetics in VeroE6 cells. (C) Viral replication kinetics in Calu3 cells. SARS-CoV-2 (0.1 TCID₅₀ of Omicron or Delta variant) was inoculated to cells for viral infection. Cell culture supernatants were collected at indicated time points for RT-qPCR assay to determine viral RNA titres. Data are presented as mean ± SD from two independent experiments with four biological samples. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001 when compared with Omicron variant.

Figure 2.

Delta variant outcompeted the replication of Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. VeroE6/TMPRSS2, VeroE6, and Calu3 cells were co-infected by the Delta and Omicron variants with the Delta:Omicron ratio of 1:10, 1:1 and 10:1. Cell culture supernatants were collected at 24 hpi (A) and 48 hpi (B) for next-generation sequencing. The percentage of reads corresponding to the Delta and Omicron variant is determined for spike amino acid residues 222 (Omicron: A; Delta: V), 655 (Omicron: Y; Delta H), and 950 (Omicron: D, Delta: N). * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001.

Figure 3.

Camostat could not potently inhibit the replication of Omicron variant in VeroE6/TMPRSS2 cells. (A) Bafilomycin A1 inhibited the Omicron and Delta replication in VeroE6/TMPRSS2 cells. (B) Chloroquine inhibited the Omicron and Delta replication in VeroE6/TMPRSS2 cells. (C) Camostat could potently inhibit Delta variant but not Omicron variant replication in VeroE6/TMPRSS2 cells. Cells were pretreated by the indicated bafilomycin A1, chloroquine or camostat before viral infection, 1000 TCID₅₀ of virus was added to cells for infection and then viral RNA copies in cell lysate were measured at 8 hpi by RT-qPCR. Viral RNA (%) was normalized to the viral RNA copy of untreated virus. ** indicates P < 0.01 and *** indicates P < 0.001 when compared with untreated virus. Data are presented as mean ± SD from three independent biological samples.

Figure 4.

SARS-CoV-2 mediated cell-cell fusion. (A) Cell GFP images of infected cells at indicated post infection time. (B) Bright field images of infected cells at indicated post infection time. VeroE6/TMPRSS2 cells were transfected with GFP plasmid and infected with 0.1 TCID₅₀ virus (Omicron and Delta variant). Images were taken at 24, 48, and 72 hpi. GFP-transfected cells without viral infection were the negative control of cell fusion (no infection). Scale bar = 50 μm. Experiment images were taken from two independent biological samples.