BMJ Open Respiratory Research (Jan 2021)

Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells

  • Hiroshi Ishii,
  • Tadaki Suzuki,
  • Souichi Yamada,
  • Shuetsu Fukushi,
  • Hitomi Kinoshita,
  • Makoto Ohnishi,
  • Tsuguto Fujimoto,
  • Masayuki Saijo,
  • Ken Maeda,
  • Nozomu Hanaoka,
  • Naomi Nojiri,
  • Ai Kawana-Tachikawa,
  • Shigeru Kusagawa,
  • Koichi Ishikawa,
  • Shigeyoshi Harada,
  • Saori Matsuoka,
  • Tadashi Kikuchi,
  • Sayuri Seki,
  • Midori Nakamura-Hoshi,
  • Shoji Miki,
  • Lucky Ronald Runtuwene,
  • Nobuo Koizumi,
  • Sunao Iyoda,
  • Hideyuki Takahashi,
  • Hidemasa Izumiya,
  • Jiro Mitobe,
  • Shouji Yamamoto,
  • Masatomo Morita,
  • Ken-ichi Lee,
  • Ken Shimuta,
  • Kyoko Saito,
  • Masayoshi Fukasawa,
  • Yasutaka Hoshino,
  • Ken Miyazawa,
  • Minoru Nagi,
  • Chikako Shimokawa,
  • Yasuyuki Morishima,
  • Takashi Sakudoh,
  • Yoshihiro Kaku,
  • Chang Kweng Lim,
  • Shigeru Tajima,
  • Takahiro Maeki,
  • Eri Nakayama,
  • Satoshi Taniguchi,
  • Motohiko Ogawa,
  • Takanobu Kato,
  • Hussein Hassan Aly,
  • Kousho Wakae,
  • Kento Fukano

DOI
https://doi.org/10.1136/bmjresp-2020-000830
Journal volume & issue
Vol. 8, no. 1

Abstract

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Background An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.Methods We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.Results The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.Conclusion In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.