Improved efficacy of SARS-CoV-2 isolation from COVID-19 clinical specimens using VeroE6 cells overexpressing TMPRSS2 and human ACE2

The cell culture-based isolation of novel coronavirus SARS-CoV-2 from clinical specimens obtained from patients with suspected COVID-19 is important not only for laboratory diagnosis but also for obtaining live virus to characterize emerging variants. Previous studies report that monkey kidney-derived VeroE6/TMPRSS2 cells allow efficient isolation of SARS-CoV-2 from clinical specimens because these cells show stable expression of the receptor molecule monkey ACE2 and the serine-protease TMPRSS2. Here, we demonstrated that VeroE6 cells overexpressing human ACE2 and TMPRSS2 (Vero E6-TMPRSS2-T2A-ACE2 cells) are superior to VeroE6/TMPRSS2 for isolating SARS-CoV-2 from clinical specimens. These cells showed a 1.6-fold increase in efficiency in SARS-CoV-2 isolation, and were particularly effective for clinical specimens with a relatively low viral load (< 10⁶ copies/mL). When using vesicular stomatitis virus (VSV) pseudoviruses (VSV/SARS-2pv) bearing the spike proteins of all of the tested SARS-CoV-2 strains, Vero E6-TMPRSS2-T2A-ACE2 cells showed a 2- to fourfold increase in infectivity. Furthermore, the results of virus titration and neutralization antibody assays using Vero E6-TMPRSS2-T2A-ACE2 cells were different from those using VeroE6/TMPRSS2, highlighting the importance of selecting appropriate cell culture systems to determine SARS-CoV-2 infectivity.