tetano
Editor, Senior Moderator
Sci Rep
. 2024 Oct 22;14(1):24858.
doi: 10.1038/s41598-024-75038-4. Improved efficacy of SARS-CoV-2 isolation from COVID-19 clinical specimens using VeroE6 cells overexpressing TMPRSS2 and human ACE2
Hitomi Kinoshita[SUP] #[/SUP][SUP] 1 [/SUP], Tsukasa Yamamoto[SUP] #[/SUP][SUP] 2 [/SUP], Yudai Kuroda[SUP] #[/SUP][SUP] 2 [/SUP], Yusuke Inoue[SUP] 2 [/SUP], Kaya Miyazaki[SUP] 3 [/SUP], Norio Ohmagari[SUP] 4 [/SUP], Daisuke Tokita[SUP] 5 [/SUP], Phu Hoang Anh Nguyen[SUP] 1 [/SUP], Souichi Yamada[SUP] 1 [/SUP], Shizuko Harada[SUP] 1 [/SUP], Takayuki Kanno[SUP] 6 [/SUP], Kenichiro Takahashi[SUP] 7 [/SUP], Masumichi Saito[SUP] 7 8 [/SUP], Kazuya Shirato[SUP] 9 [/SUP], Ikuyo Takayama[SUP] 3 [/SUP], Shinji Watanabe[SUP] 3 [/SUP], Tomoya Saito[SUP] 7 [/SUP], Hideki Ebihara[SUP] 1 [/SUP], Tadaki Suzuki[SUP] 6 [/SUP], Ken Maeda[SUP] 2 [/SUP], Shuetsu Fukushi[SUP] 10 [/SUP]
Affiliations
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 (< 10[SUP]6[/SUP] 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.
Keywords: ACE2; SARS-CoV-2; TMPRSS2; Virus isolation.
. 2024 Oct 22;14(1):24858.
doi: 10.1038/s41598-024-75038-4. Improved efficacy of SARS-CoV-2 isolation from COVID-19 clinical specimens using VeroE6 cells overexpressing TMPRSS2 and human ACE2
Hitomi Kinoshita[SUP] #[/SUP][SUP] 1 [/SUP], Tsukasa Yamamoto[SUP] #[/SUP][SUP] 2 [/SUP], Yudai Kuroda[SUP] #[/SUP][SUP] 2 [/SUP], Yusuke Inoue[SUP] 2 [/SUP], Kaya Miyazaki[SUP] 3 [/SUP], Norio Ohmagari[SUP] 4 [/SUP], Daisuke Tokita[SUP] 5 [/SUP], Phu Hoang Anh Nguyen[SUP] 1 [/SUP], Souichi Yamada[SUP] 1 [/SUP], Shizuko Harada[SUP] 1 [/SUP], Takayuki Kanno[SUP] 6 [/SUP], Kenichiro Takahashi[SUP] 7 [/SUP], Masumichi Saito[SUP] 7 8 [/SUP], Kazuya Shirato[SUP] 9 [/SUP], Ikuyo Takayama[SUP] 3 [/SUP], Shinji Watanabe[SUP] 3 [/SUP], Tomoya Saito[SUP] 7 [/SUP], Hideki Ebihara[SUP] 1 [/SUP], Tadaki Suzuki[SUP] 6 [/SUP], Ken Maeda[SUP] 2 [/SUP], Shuetsu Fukushi[SUP] 10 [/SUP]
Affiliations
- PMID: 39438626
- PMCID: PMC11496680
- DOI: 10.1038/s41598-024-75038-4
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 (< 10[SUP]6[/SUP] 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.
Keywords: ACE2; SARS-CoV-2; TMPRSS2; Virus isolation.