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

• Published in: BMJ open respiratory research Vol. 8; no. 1; p. e000830
• Main Authors: Yamada, Souichi, Fukushi, Shuetsu, Kinoshita, Hitomi, Ohnishi, Makoto, Suzuki, Tadaki, Fujimoto, Tsuguto, Saijo, Masayuki, Maeda, Ken, Hanaoka, Nozomu, Nojiri, Naomi, Kawana-Tachikawa, Ai, Kusagawa, Shigeru, Ishikawa, Koichi, Harada, Shigeyoshi, Matsuoka, Saori, Kikuchi, Tadashi, Ishii, Hiroshi, Seki, Sayuri, Nakamura-Hoshi, Midori, Miki, Shoji, Runtuwene, Lucky Ronald, Koizumi, Nobuo, Iyoda, Sunao, Takahashi, Hideyuki, Izumiya, Hidemasa, Mitobe, Jiro, Yamamoto, Shouji, Morita, Masatomo, Lee, Ken-ichi, Shimuta, Ken, Saito, Kyoko, Fukasawa, Masayoshi, Hoshino, Yasutaka, Miyazawa, Ken, Nagi, Minoru, Shimokawa, Chikako, Morishima, Yasuyuki, Sakudoh, Takashi, Kaku, Yoshihiro, Lim, Chang Kweng, Tajima, Shigeru, Maeki, Takahiro, Nakayama, Eri, Taniguchi, Satoshi, Ogawa, Motohiko, Kato, Takanobu, Aly, Hussein Hassan, Wakae, Kousho, Fukano, Kento
• Format: Journal Article
• Language: English
• Published: England British Thoracic Society 01.02.2021; BMJ Publishing Group LTD; BMJ Publishing Group
• Subjects: Animals; Cell culture; Cell Line; Chlorocebus aethiops; Coronaviruses; COVID-19; COVID-19 - transmission; COVID-19 Nucleic Acid Testing; Cytopathogenic Effect, Viral; Disease transmission; Humans; Laboratories; Nasal Cavity - virology; Nasopharynx - virology; Public health; Respiratory Infection; Saliva - virology; SARS-CoV-2 - isolation & purification; SARS-CoV-2 - pathogenicity; Serine Endopeptidases - genetics; Severe acute respiratory syndrome coronavirus 2; Specimen Handling; Tomography; Vero Cells; viral infection; Viral infections; Viruses; Japan; COVID-19; viral infection
• ISSN: 2052-4439; 2052-4439
• DOI: 10.1136/bmjresp-2020-000830

BackgroundAn 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.MethodsWe 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.ResultsThe 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.ConclusionIn 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.