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

• Published in: Scientific reports Vol. 14; no. 1; pp. 24858 - 12
• Main Authors: Kinoshita, Hitomi, Yamamoto, Tsukasa, Kuroda, Yudai, Inoue, Yusuke, Miyazaki, Kaya, Ohmagari, Norio, Tokita, Daisuke, Nguyen, Phu Hoang Anh, Yamada, Souichi, Harada, Shizuko, Kanno, Takayuki, Takahashi, Kenichiro, Saito, Masumichi, Shirato, Kazuya, Takayama, Ikuyo, Watanabe, Shinji, Saito, Tomoya, Ebihara, Hideki, Suzuki, Tadaki, Maeda, Ken, Fukushi, Shuetsu
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22.10.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/326/596; 631/326/596/4130; ACE2; Angiotensin-converting enzyme 2; Angiotensin-Converting Enzyme 2 - genetics; Angiotensin-Converting Enzyme 2 - metabolism; Animals; Cell culture; Chlorocebus aethiops; Coronaviruses; COVID-19; COVID-19 - metabolism; COVID-19 - virology; Humanities and Social Sciences; Humans; Infectivity; multidisciplinary; Neutralization; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; SARS-CoV-2 - metabolism; Science; Science (multidisciplinary); Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Severe acute respiratory syndrome coronavirus 2; Stomatitis; Titration; TMPRSS2; Vero Cells; Viral Load; Virus isolation; ACE2; TMPRSS2; SARS-CoV-2; Virus isolation
• ISSN: 2045-2322; 2045-2322
• 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 6 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.