Ambecovirus, a novel Betacoronavirus subgenus circulating in neotropical bats, sheds new light on bat-borne coronaviruses evolution.

Understanding the viral diversity harboured by wildlife is essential for effective mapping and prevention of future zoonotic outbreaks. Bats, in particular, are recognized as natural reservoirs for several high-impact zoonotic viral pathogens, including coronaviruses responsible for Severe Acute Respiratory Syndrome (SARS), the rabies virus, diverse paramyxoviruses, Marburg, Ebola, Nipah, and Hendra viruses. However, a large extent of bat viruses remains unexplored, especially in highly biodiverse regions of the Neotropics such as Brazilian ecosystems. We used a meta-transcriptomic approach to characterize new virus genomes found in blood, oral, and anal samples collected from cave- and noncave bats from Northeast Brazil, Caatinga, and Atlantic Forest biomes. From a total of 19 coronavirus-positive bats, we have assembled two complete genomes of a new Betacoronavirus subgenus, named Ambecovirus (American betacoronavirus). The subgenus herein described is phylogenetically placed between the Sarbeco-/Hibeco-/Nobecovirus and the Merbeco-/Embecovirus clades, being basal to the former. While the conserved S2 region of the spike protein retained hallmark domains, including HR1 and HR2, the S1/S2 cleavage site and the furin cleavage site, the S1 region consistently displayed only the N-terminal domain. The receptor-binding domain from the C-terminal domai (CTD) region could not be identified due to high dissimilarity relative to known congeners. The detection of Ambercovirus in sympatric Pteronotus gymnonotus and Carollia perspicillata bats suggests potential interspecies transmission. Longitudinal sampling confirmed persistent Ambecovirus infection in P. gymnonotus over multiple years and virus dispersion at a minimum distance of 270 km between caves. The present study confirms that viral diversity in neotropical hosts remains largely unknown, not just in Brazil but likely in the other countries of the region, supporting the need for a systematic approach to virome exploration and analysis followed by in vitro experimentation to assess zoonotic potential.