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PreprintCited by 2Animal Virus Infections StudiesZoonotic diseases and public healthSARS-CoV-2 and COVID-19 Research

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

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da Luz Wallau, Gabriel, Barbier, Eder, Machado, Lais Ceschini, da Silva, Alexandre Freitas, Dias, Yago Jose Mariz, Tomazatos, Alexandru, Horváth, Balázs, Lins, Roberto D., Bernard, Enrico, Cadar, Dániel·2025·Virus Evolution
DOI 10.1101/2025.07.20.665586PMC PMC12765449

Preprint — published as 10.1093/ve/veaf094 · Paper page

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Abstract

Understanding the viral diversity harboured by wildlife is essential for effective prediction and prevention of future zoonotic outbreaks. Bats, in particular, are recognized as natural reservoirs for several zoonotic viral pathogens of high impact on public health, including coronaviruses responsible for SARS, the rabies virus, Marburg, Ebola, Nipah and Hendra viruses. However, the large extent of bat viruses remains unexplored, especially in highly biodiverse regions of the Neotropics such as Brazil. We used a meta-transcriptomic to characterize new virus genomes found in blood, oral and anal samples collected from cave- and non-cave bats from Northeast Brazil. 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 -/ Nebeco virus 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 could not be identified due to high dissimilarity relative to known congeneres. The detection of Ambercovirus in sympatric Pteronotus gymnonotus and Carollia perspicillata bats suggests 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.

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Figure 1 Bat sampling performed during the study period. (A) Sampling sites including 11 caves (circles) and 1 urban (triangle) site in the Northeast part of Brazil. Colours denote two of the six Brazilian biomes; orange (Caatinga) and green (Atlantic Forest). (B) Number of specimens collected per sampling site during this study. The first two letters of the site acronym stands for the name of the cave/site (GS, Gruta do Sobradinho; BL, Boqueirão de Lavras II; GF, Gruta do Farias; FF, Furna Feia; FU, Furna do Urubu; GA, Gruta do Arnoud; MR, Meu Rei; FM, Furna do Morcego; FG, Furna do Gato; RE, Recife; UR, Urubu; CP, Casa de Pedra) while the following two letters stand for the state where the cave was located (CE, Ceará; RN, Rio Grande do Norte; PE, Pernambuco; and SE, Sergipe).
Figure 1 Bat sampling performed during the study period. (A) Sampling sites including 11 caves (circles) and 1 urban (triangle) site in the Northeast part of Brazil. Colours denote two of the six Brazilian biomes; orange (Caatinga) and green (Atlantic Forest). (B) Number of specimens collected per sampling site during this study. The first two letters of the site acronym stands for the name of the cave/site (GS, Gruta do Sobradinho; BL, Boqueirão de Lavras II; GF, Gruta do Farias; FF, Furna Feia; FU, Furna do Urubu; GA, Gruta do Arnoud; MR, Meu Rei; FM, Furna do Morcego; FG, Furna do Gato; RE, Recife; UR, Urubu; CP, Casa de Pedra) while the following two letters stand for the state where the cave was located (CE, Ceará; RN, Rio Grande do Norte; PE, Pernambuco; and SE, Sergipe).CC BY · PubMed Central
Figure 2 Genomic features and spike protein domain annotation of the Ambecovirus full genome recovered in this study. (A) Coverage breadth, depth, and complete genomic annotation of the new betacoronavirus genome; (B) schematic of the spike protein highlighting the S1 and S2 subunits, including conserved domains; (C) amino acid alignment of the Ambecovirus spike protein against the top 100 BLASTp hits, illustrating divergence in the S1 region; (D) amino acid alignment of the S2 domains found in the CDD search including the spike protein of the two complete genomes obtained in this study and reference coronavirus genomes.
Figure 2 Genomic features and spike protein domain annotation of the Ambecovirus full genome recovered in this study. (A) Coverage breadth, depth, and complete genomic annotation of the new betacoronavirus genome; (B) schematic of the spike protein highlighting the S1 and S2 subunits, including conserved domains; (C) amino acid alignment of the Ambecovirus spike protein against the top 100 BLASTp hits, illustrating divergence in the S1 region; (D) amino acid alignment of the S2 domains found in the CDD search including the spike protein of the two complete genomes obtained in this study and reference coronavirus genomes.CC BY · PubMed Central
Figure 3 Superposition of a SwissModel predicted structure (orange) of the spike protein from ambecov based on the PDB ID 9BSW template from HCoV-HKU1 (cyan) depicted in cartoon model, and comparison of the electrostatic surface potential for the NTD region. (A) shows superposition for the spike protein in its trimeric form. (B) shows the superposition for the NTDs, highlighting the predicted β-sheet motif for the model (orange). (C) shows the superposition for the predicted CTD region. The putative CTD region for the model (orange) is shown as highly unstructured, the result of a massive three-dimensional misalignment outside of the core region due to a low sequence identity between template and model. (D) shows similarity in the electrostatic surface potential for sialic acid binding β-sheet groove in the NTD from HCoV-HKU1 (left) and the corresponding region in the ambecov model (right) encompassed within thin orange lines (electrostatic potential ranges from −5kT/e (red) to +5 kT/e (blue) and it is plotted onto the van der Waals surface).
Figure 3 Superposition of a SwissModel predicted structure (orange) of the spike protein from ambecov based on the PDB ID 9BSW template from HCoV-HKU1 (cyan) depicted in cartoon model, and comparison of the electrostatic surface potential for the NTD region. (A) shows superposition for the spike protein in its trimeric form. (B) shows the superposition for the NTDs, highlighting the predicted β-sheet motif for the model (orange). (C) shows the superposition for the predicted CTD region. The putative CTD region for the model (orange) is shown as highly unstructured, the result of a massive three-dimensional misalignment outside of the core region due to a low sequence identity between template and model. (D) shows similarity in the electrostatic surface potential for sialic acid binding β-sheet groove in the NTD from HCoV-HKU1 (left) and the corresponding region in the ambecov model (right) encompassed within thin orange lines (electrostatic potential ranges from −5kT/e (red) to +5 kT/e (blue) and it is plotted onto the van der Waals surface).CC BY · PubMed Central
Figure 4 Phylogenetic reconstruction of the two complete coronavirus genomes characterized in this study and representative reference coronavirus genomes at the genus level. (A) Maximum likelihood phylogenetic reconstruction based on amino acid sequences of Nidovirales conserved domains (3CLpro, NiRAN, RdRP, ZBD, and HEL1 domains) (Supplementary File 4). (B) Total evidence nucleotide tree based on the RdRp coding region including partial (439 bp) and full RdRp nucleotide coding region (~2790 bp) of the coronaviruses sequenced in this study (Supplementary File 5). Branch support values above nodes were calculated by aLRT/ultrafast bootstrap and posterior probability. Tip colours denote the country of sampling.
Figure 4 Phylogenetic reconstruction of the two complete coronavirus genomes characterized in this study and representative reference coronavirus genomes at the genus level. (A) Maximum likelihood phylogenetic reconstruction based on amino acid sequences of Nidovirales conserved domains (3CLpro, NiRAN, RdRP, ZBD, and HEL1 domains) (Supplementary File 4). (B) Total evidence nucleotide tree based on the RdRp coding region including partial (439 bp) and full RdRp nucleotide coding region (~2790 bp) of the coronaviruses sequenced in this study (Supplementary File 5). Branch support values above nodes were calculated by aLRT/ultrafast bootstrap and posterior probability. Tip colours denote the country of sampling.CC BY · PubMed Central
Figure 5 Spatio-temporal distribution of ambecovirus-positive samples obtained from P. gymnonotus and C. perspicillata. (A) Map of the caves and urban sampling sites visited along the study showing coloured caves where P. gymnonotus, C. perspicillata, or both species co-occur. (B) Number of individual samples per cave during the study period, including the number of partial or complete genomes recovered from metagenomic data.
Figure 5 Spatio-temporal distribution of ambecovirus-positive samples obtained from P. gymnonotus and C. perspicillata. (A) Map of the caves and urban sampling sites visited along the study showing coloured caves where P. gymnonotus, C. perspicillata, or both species co-occur. (B) Number of individual samples per cave during the study period, including the number of partial or complete genomes recovered from metagenomic data.CC BY · PubMed Central

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Metadata

Authors
da Luz Wallau, G., Barbier, E., Machado, L. C., da Silva, A. F., Dias, Y. J. M., Tomazatos, A., Horváth, B., Lins, R. D., Bernard, E., & Cadar, D.
Affiliations
  • Universidade Federal de Pernambuco (BR)
  • Instituto Aggeu Magalhães (BR)
  • Bernhard Nocht Institute for Tropical Medicine (DE)
  • Universidade Federal de Lavras (BR)
Corresponding author
Cadar, Dániel
ORCID
Venue
Virus Evolution
Publisher
openRxiv
Published
2025-07-23
Volume
11
Issue
1
Pages
veaf094-veaf094
ISSN
2057-1577 (ISSN-L)
DOI
10.1101/2025.07.20.665586
PMC
PMC12765449
OpenAlex
W4417086166
Cited by
2
References
1
Citations by year
2026: 2
FWCI
1.23
Type
journal article
Language
en
Open access
Licence
Locations
  • Virus Evolution (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Arca - Repositório Institucional da Fiocruz (submittedVersion, other-oa)
Funders
  • Alexander von Humboldt Foundation
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico
Topics
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Zoonotic diseases and public health
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
Keywords
Neotropical bats, Pteronotus gymnonotus, Carollia perspicillata, viral evolution, spike protein, interspecies transmission, viral persistence, zoonotic potential, cave-dwelling bats, viral metatranscriptomics, Atlantic Forest, Caatinga

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