key: cord-0330699-k7jcxx3p
authors: Murakami, Shin; Kitamura, Tomoya; Matsugo, Hiromichi; Kamiki, Haruhiko; Oyabu, Ken; Sekine, Wataru; Takenaka-Uema, Akiko; Sakai-Tagawa, Yuko; Kawaoka, Yoshihiro; Horimoto, Taisuke
title: Isolation of bat sarbecoviruses of SARS-CoV-2 clade, Japan
date: 2022-05-16
journal: bioRxiv
DOI: 10.1101/2022.05.16.492045
sha: 973297693abc47b4e7e98e9640ef038887d94985
doc_id: 330699
cord_uid: k7jcxx3p

Betacoronaviruses have caused 3 outbreaks in the past 2 decades. SARS-CoV-2, in particular, has caused a serious pandemic. As the betacoronaviruses are considered to originate from bats, surveillance of bat betacoronaviruses is crucial for understanding the mechanism of cross-species transition and potential for future outbreaks. We previously detected and characterized a SARS-CoV-2-related sarbecovirus, Rc-o319, from Rhinolophus cornutus in Japan. Here, we detected several bat sarbecoviruses of the SARS-CoV-2 clade from R. cornutus in multiple locations in Japan, and successfully isolated them using Vero/TMPRSS2 cells stably expressing R. cornutus ACE2 (Vero-RcACE2). The coding sequences of S1 region varied among isolates, whereas other genetic regions were highly conserved. Isolates were efficiently grown in Vero-RcACE2 cells, but did not replicate in Vero/TMPRSS2 cells stably expressing human ACE2, suggesting a narrow host range. Further long-term epidemiological studies of sarbecoviruses in wildlife are expected to facilitate the assessment of the risk of their spillover potential.

Diluents were inoculated into 6-well plates containing Vero-RcACE2 cells, and plates were incubated for 60 min at 37 °C after removing the inoculum. Wells were then 1 4 0 washed once with cell maintenance medium, another 2 mL of cell maintenance medium Rc-mk2, and Rc-kw8 have been deposited in GenBank (accession Nos. LC663958, 1 5 8 LC663959, and LC663793, respectively). The nucleotide sequences of sarbecoviruses were aligned using ClustalW version 2.1 1 6 2 (Clustal, https://www.clustal.org). Phylogenetic trees were then constructed by allowed for 1 h for viral adsorption. After removing the inocula, cells were incubated in 1 6 9 cell maintenance medium, and the supernatants were collected at 12 h intervals. Viral 1 7 0 titers were measured using a plaque assay, in which cells inoculated with diluted viruses 1 7 1 were overlaid and incubated with DMEM containing 1 % agarose and 1 % FCS for 2 d, followed by staining with crystal violet before counting plaques. We collected fecal samples from bats belonging to the Rhinolophus cornutus and sarbecovirus in 1 or 2 R. cornutus samples in each prefecture (Table 1 ). In contrast, all R. In our previous study, we showed that a VSV-based pseudotyped virus possessing the S clade. In addition, the Japanese bat isolates were unable to use hACE2 as a cell entry of the mechanism and the potential of bat sarbecoviruses to overcome host barriers.

Because of the pilot study aspect of this study, the number of samples and the 2 5 0 bat species studied were limited. Four species of Rhinolophus bats inhabit Japan: R. widely distributed, exhibiting a major population in Japan, whereas the latter 2 species 2 5 3 are mainly confined in Okinawa prefecture, which is located far southwest of the main 2 5 4 island of Japan. In this study, the bat sarbecoviruses were isolated from R. cornutus but 2 5 5 not from R. ferrumequinum. Since the virus has been detected from R. ferrumequinum in China (5), it is expected that increasing the number of samples will clarify whether R. surveillance of bat coronaviruses in Korea: Diversity and distribution pattern. Dec;12(1):6563. Aug;584(7821):450-6. Phylogenetic tree of bat sarbecoviruses was generated using the full genome nucleotide Rc-o319, Rc-os20, Rc-mk2, and Rc-kw8 or SARS-CoV-2 (B.1.1.7) were inoculated into 

Rc-mk2 Rc-kw8 

Rc-os20 (Niigata) Rc-kw8 (Shizuoka)

Bat sarbecovirus Rm1 DQ412043 Bat sarbecovirus HKU3-1 DQ022305 Bat sarbecovirus Rf1 DQ412042 Bat sarbecovirus 16BO133 KY938558 Bat sarbecovirus Rp3 DQ071615 Bat sarbecovirus Rf4092 KY417145 SARS-CoV

Bat sarbecovirus Rs3367 KC881006 Bat sarbecovirus RsSHC014 KC881005 Bat sarbecovirus BM48-31 NC 014470 Bat sarbecovirus BtKY72 KY352407 SARS-CoV-2 WHU01 MN988668 Bat sarbecovirus BANAL-20-52 MZ937000 Bat sarbecovirus RaTG13 MN996532 Bat sarbecovirus RacCS203 MW251308 Bat sarbecovirus RmYN02 EPI_ISL_412977 Bat sarbecovirus RShSTT182 EPI_ISL_852604 Pangolin sarbecovirus Guangdong/1/2019 EPI_ISL_410721 Bat sarbecovirus bat-SL-CoVZXC21 MG772934 Bat sarbecovirus bat-SL-CoVZC45 MG772933

Pangolin sarbecovirus Guangxi/P4L/2017 EPI_ISL_410538 Bat sarbecovirus Ra7909 OL674077 Bat sarbecovirus Rc-mk2 LC663959 Bat sarbecovirus Rc-os20 LC663958 Bat sarbecovirus Rc-o319 LC556375 Bat sarbecovirus Rc-kw8 LC663793

Rousettus Bat nobecovirus GCCDC1 356 KU762338 MERS-CoV EMC/2012 JX869059 Human coronavirus OC43 AY391777 Human coronavirus HKU1 MK167038

Martin DP, Varsani A, Roumagnac P, Botha G, Maslamoney S, Schwab T, et al. Broad neutralization of SARS-related viruses by human monoclonal antibodies. Science. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.