Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture

• Published in: Japanese Journal of Infectious Diseases Vol. 74; no. 4; pp. 285 - 292
• Main Authors: Komabayashi, Kenichi, Matoba, Yohei, Seto, Junji, Ikeda, Yoko, Tanaka, Waka, Aoki, Yoko, Ikeda, Tatsuya, Matsuzaki, Yoko, Itagaki, Tsutomu, Shirato, Kazuya, Mizuta, Katsumi
• Format: Journal Article
• Language: English
• Published: Tokyo National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee 31.07.2021; Japan Science and Technology Agency
• Subjects: ACE2; air-liquid interface culture; Angiotensin-converting enzyme 2; Cell culture; Cell lines; common cold; Coronaviruses; COVID-19; Cryopreservation; Culture; Epithelium; Genomes; primary culture; Respiratory tract; SARS-CoV-2; seasonal CoV; Severe acute respiratory syndrome coronavirus 2; Virions; Japan
• ISSN: 1344-6304; 1884-2836; 1884-2836
• DOI: 10.7883/yoken.JJID.2020.776

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.