Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells
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 transmiss...
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BMJ Publishing Group
2021-01-01
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| Series: | BMJ Open Respiratory Research |
| Online Access: | https://bmjopenrespres.bmj.com/content/8/1/e000830.full |
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| author | 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 |
| author_facet | 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 |
| author_sort | Hiroshi Ishii |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b9d2ad04ff3d4af5a4c6a118be36fca9 |
| institution | Kabale University |
| issn | 2052-4439 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | BMJ Open Respiratory Research |
| spelling | doaj-art-b9d2ad04ff3d4af5a4c6a118be36fca92024-11-24T10:05:10ZengBMJ Publishing GroupBMJ Open Respiratory Research2052-44392021-01-018110.1136/bmjresp-2020-000830Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cellsHiroshi Ishii0Tadaki Suzuki1Souichi Yamada2Shuetsu Fukushi3Hitomi Kinoshita4Makoto Ohnishi5Tsuguto Fujimoto6Masayuki Saijo7Ken Maeda8Nozomu HanaokaNaomi NojiriAi Kawana-TachikawaShigeru KusagawaKoichi IshikawaShigeyoshi HaradaSaori MatsuokaTadashi KikuchiSayuri SekiMidori Nakamura-HoshiShoji MikiLucky Ronald RuntuweneNobuo KoizumiSunao IyodaHideyuki Takahashi9Hidemasa IzumiyaJiro MitobeShouji YamamotoMasatomo MoritaKen-ichi LeeKen ShimutaKyoko SaitoMasayoshi FukasawaYasutaka HoshinoKen MiyazawaMinoru NagiChikako ShimokawaYasuyuki MorishimaTakashi SakudohYoshihiro KakuChang Kweng LimShigeru TajimaTakahiro MaekiEri NakayamaSatoshi TaniguchiMotohiko OgawaTakanobu KatoHussein Hassan AlyKousho WakaeKento FukanoDepartment of Respiratory Medicine, Fukuoka University Chikushi Hospital, Chikushino, Fukuoka, Japan3 Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, JapanVirology 1, NIID, Shinjuku-ku, Tokyo, JapanVirology 1, NIID, Shinjuku-ku, Tokyo, JapanVirology 1, NIID, Shinjuku-ku, Tokyo, JapanNIID, Shinjuku-ku, Tokyo, JapanCenter for Infectious Disease Risk Management, NIID, Shinjuku-ku, Tokyo, JapanVirology 1, NIID, Shinjuku-ku, Tokyo, JapanDepartment of Veterinary Science, NIID, Shinjuku-ku, Tokyo, JapanTokyo metropolitan cancer and disease center Komagome hospital, Department of systemic immunological disease, Tokyo, Bunkyoku, JapanBackground 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.https://bmjopenrespres.bmj.com/content/8/1/e000830.full |
| spellingShingle | 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 Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells BMJ Open Respiratory Research |
| title | Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells |
| title_full | Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells |
| title_fullStr | Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells |
| title_full_unstemmed | Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells |
| title_short | Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells |
| title_sort | assessment of sars cov 2 infectivity of upper respiratory specimens from covid 19 patients by virus isolation using veroe6 tmprss2 cells |
| url | https://bmjopenrespres.bmj.com/content/8/1/e000830.full |
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