D uring the past 20 years, coronaviruses belonging to the genus Betacoronavirus have caused multiple human epidemic or pandemic diseases, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease (COVID-19). Two viruses of the subgenus Sarbecovirus are severe acute respiratory syndrome coronavirus (SARS-CoV), which causes SARS, and SARS-CoV-2, which causes COVID-19. Although Rhinolophus spp. bats in Asia, Europe, and Africa are considered natural reservoirs of sarbecoviruses (1-3), the epidemiology and distribution of these viruses remain largely unknown, especially outside China. Previously, partial RNA-dependent RNA polymerase (RdRp) genes of betacoronaviruses were detected in little Japanese horseshoe bats (Rhinolophus cornutus) (4). However, limited sequence information left the genetic and virological properties unclear. We detected and determined the entire genome sequence of a bat sarbecovirus belonging to a phylogenetic clade that includes SARS-CoV-2 from R. cornutus bats in Japan. Further, we used a pseudotyped virus system to characterize an entry step of this virus into cells. The Study R. cornutus is an endemic bat species in Japan and is found nationwide. These bats primarily inhabit caves and abandoned tunnels in the countryside during daytime and capture insects at night outside their roosts. R. cornutus bats often cohabit with other insectivorous bats, such as R. ferrumequinum or Myotis macrodactylus, and occasionally with wild animals, such as the masked palm civet (Paguma larvata), in their daytime roosts. In 2013, we captured 4 R. cornutus bats in a cave in Iwate prefecture, Japan, and extracted RNA from fresh feces. Then, we used real-time reverse transcription PCR (rRT-PCR) to detect the partial RdRp gene of sarbecovirus from 2 samples by using a pair of primers designed to detect betacoronavirus. In 2020, we performed RNA sequencing and determined the full genome sequence of 1 sample, Rc-o319, which exhibited lower cycle threshold value by rRT-PCR. We performed a BLAST (https://blast.ncbi.nlm. nih.gov/Blast.cgi) analysis of the full genome of Rc-o319, which showed Rc-o319 had the highest nucleotide homology to SARS-CoV-2 HKG/HKU-904a/2020 strain (GenBank accession no. MT365032) with a query cover of 96% and sequence identity of 81.47%. The maximum-likelihood analysis with sarbecoviruses demonstrated that the full genome and spike protein (S) gene of Rc-o319 were positioned within a specific clade that included SARS-CoV-2 (Figure 1, panels A, B). Amino acid sequences of open reading frame 1ab (ORF1ab) and S of Rc-o319 also were positioned within the SARS-CoV-2 clade (Figure 1 panels C and D). The phylogenetic trees maintained the same topology between ORF1ab and S, indicating that no recombination event occurred in Rc-0319, which was supported by similarity plot analysis (Appendix Fig