Abstract
BACKGROUND: Bat-derived sarbecoviruses, members of the Betacoronavirus genus, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose a zoonotic threat. While sarbecoviruses have been isolated from bats in eastern Japan, their distribution and diversity in southern Japan remain poorly understood. We investigated the genetic characteristics and zoonotic potential of bat-derived sarbecoviruses from Rhinolophus cornutus in the Kagoshima and Kumamoto Prefectures, Japan. METHODS: We collected 133 oral swab samples from R. cornutus between 2022 and 2025. The samples were inoculated into various Vero cell derivatives to isolate sarbecoviruses. The virus genomes were sequenced and subjected to phylogenetic analyses. Viral replication was assessed in vitro. Cross-species potential was evaluated in vivo through experimental infection in Syrian hamsters. RESULTS: Six and three sarbecoviruses from the Kagoshima and Kumamoto Prefectures, respectively, were isolated. Genetic analyses revealed substantial diversity among our isolates (95.98–99.95% nucleotide identity). Phylogenetic analyses revealed clustering of the isolates separately from those found in eastern Japan, indicating regional genetic divergence among bat populations. Our isolates exhibited deletions in the receptor-binding domain of the spike protein. These deletions attenuate the viral binding affinity to human angiotensin-converting enzyme 2 (ACE2) receptors. This suggests a limited capacity of our isolates to bind human ACE2. Our isolates exclusively replicated in Vero cells expressing R. cornutus-derived ACE2 (RcACE2). Syrian hamsters, a model for SARS-CoV-2 infection, were not susceptible to infection from our isolates, suggesting limited potential for cross-species transmission. CONCLUSIONS: We characterized genetically diverse sarbecoviruses circulating in R. cornutus populations in southern Japan. Although the isolates appear to have a low immediate zoonotic risk, they likely represent only a small fraction of the sarbecoviruses circulating among bats. Given the potential presence of viruses with greater zoonotic capacity, continuous surveillance is critical for early detection of emerging threats and advancing our understanding of coronavirus ecology in bat populations.