Assessment of the zoonotic spillover potential of SARS-CoV-2-related coronaviruses from Rhinolophus pusillus bats in Thailand

Proactive surveillance of wildlife has the potential to help prevent pandemics caused by coronaviruses spilling over into human populations, but the relative value of this approach versus early outbreak investigation has been debated. This study investigated bat coronavirus zoonotic potential in a high human-wildlife interaction site in Ratchaburi, Thailand. We sampled 261 Rhinolophus pusillus bats, a known reservoir for sarbecoviruses, and detected diverse alpha- and betacoronaviruses (prevalence = 16.47%), including SARS-CoV-2-related coronaviruses, alongside paramyxoviruses at a lower prevalence (5.36%). Notably, the detected paramyxoviruses were phylogenetically distinct from previously described paramyxoviruses, suggesting the presence of previously uncharacterized viral diversity. Five complete SARS-CoV-2-related coronavirus genomes were obtained, showing significant divergence in spike and ORF8 regions and diverse recombination patterns. One identified SARS-CoV-2-related coronavirus, RpuRB21065, exhibited high spike and receptor-binding domain similarities to SARS-CoV-2. The receptor-binding domain demonstrated binding to human ACE2 and the spike protein induced syncytia in vitro. However, human sera from SARS-CoV-2 vaccinated individuals effectively neutralized RpuRB21065, suggesting pre-existing SARS-CoV-2 immunity may minimize current spillover potential. Our study is a proof-of-concept that proactive viral discovery and wildlife surveillance in EID hotspots can provide information of direct public health relevance, even in the absence of viral isolation. This study investigates the zoonotic potential of bat coronaviruses at a human-wildlife interface in Thailand. Molecular analyses identify a SARS-CoV-2-related strain capable of human ACE2 binding, which is neutralized by post-vaccination immunity.