Characterization of a MERS-related betacoronavirus in Danish brown long-eared bats (Plecotus auritus)

Abstract

Background: Bats are recognized as natural reservoir hosts for numerous viruses and are believed to be the evolutionary origin of alpha- and beta-coronaviruses (CoVs), such as SARS-CoV, SARS-CoV-2, and possibly MERS-CoV. MERS-related beta-CoVs have been identified in bat species from Africa, America, Asia, and Europe. In this study, we describe the first detection and characterization of a MERS-related beta-CoV in Danish brown long-eared bats (Plecotus auritus).

Methods: Fecal samples collected through a national surveillance program were screened using pan-CoV RT-qPCRs. Positive samples underwent ORF1b sequencing, microarray analysis and Illumina MiSeq sequencing, followed by metagenomic assembly of full-length genomes. A global phylogenetic tree was used to determine placement within the Coronaviridae family and local maximum likelihood phylogenetic analysis clarified subgroup placement. The receptor-binding potential of the spike protein to human DPP4, ACE2, and bat ACE2 orthologs was assessed through phylogenetic analysis of the receptor-binding domain (RBD), alongside homology modeling and structural analysis.

Results: Three samples tested positive for CoVs. One sample from a Soprano pipistrelle (Pipistrellus pygmaeus) was identified as alpha-CoV by ORF1b sequencing. The remaining two samples, obtained from a colony of Plecotus auritus, were identified as beta-CoVs, and separate microarray results indicated the presence of a MERS-related CoV. Full genomes were successfully assembled using a metagenomic approach. Phylogenetic analysis placed them within the merbecoviruses, forming a distinct clade with viruses detected in Vespertilionidae bats from Western Europe and East Asia. Analysis of the RBD placed them within the HKU25 clade. Structural modeling suggested hydrogen bonding patterns between the RBD and human/bat ACE2 orthologs or human DPP4, similar to known in vitro complexes, indicating potential receptor binding.

Conclusion: This is the first report of MERS-related beta-CoVs in bats from Denmark. Phylogenetic analyses reveal that these novel viruses belong to the HKU25 clade, a clade with known ACE2 receptor preference. Experimental validation is needed to confirm the receptor-binding potential, as additional interactions at the RBD-receptor interface may differ from previously described bat-merbecoviruses. Continued surveillance is crucial to identify potential intermediate hosts and assess interspecies transmission risk, with focus on the spike protein, receptor specificity, and binding affinity.

Keywords: Bats; Betacoronavirus; Denmark; Full genome sequencing; MERS-related; Merbecovirus; Molecular analysis; Phylogenetic analysis.

Fig. 1

Location of the sampling sites which were positive in the pan-CoV assays in 2020. Location of the brown long-eared bat (Plecotus auritus) sampling site at Almindingen on Bornholm in Denmark (red) and the soprano pipistrelle bat (Pipistrellus pygmaeus) sampling site at Svenstrup Gods, on Zealand in Denmark (blue)

Fig. 2

Coverage profile and genome arrangement of BtCoV/16227–9/P.aur/DK/2020, a bat MERS-related beta-CoV from a fecal sample obtained from a Plecotus auritus bat in Denmark

Fig. 3

Rooted local maximum-likelihood tree of full-length nucleotide sequences of merbecoviruses with the novel MERS-related beta-CoVs obtained from Danish Plecotus auritus bats in blue and outgroup in grey. Branch support is categorized as strong (SH-aLRT ≥ 80 and UFBoot ≥ 95), moderate (SH-aLRT ≥ 60 and UFBoot ≥ 60), and weak (below these thresholds). Phylogenetic inference was performed in IQ-Tree using the GTR + G substitution model with 1000 replicates for both SH-aLRT and UFBoot

Fig. 4

Rooted local maximum-likelihood tree showing the relationship of RBD amino acid sequences. The RBD is defined by the two glycine residues at aa position 372 and 610 in the human MERS reference sequence (JX869059). BtCoV/16227–9/P.aur/DK/2020 in blue and embecovirus outgroup in grey. Lineages in column 1 and receptor usage in column 2. Branch support is categorized as strong (SH-aLRT ≥ 80 and UFBoot ≥ 95), moderate (SH-aLRT ≥ 60 and UFBoot ≥ 60), and weak (below these thresholds). Phylogenetic inference was performed in IQ-Tree using the WAG + G4 substitution model with 1000 replicates for both SH-aLRT and UFBoot

Fig. 5

Hydrogen bonding analysis between ACE2 orthologs or DPP4 and BtCoV/16227–9/P.aur/DK/2020 spike receptor-binding domain. Homology models were produced with SWISS-MODEL using appropriate templates and hydrogen bonds (bright green) at the RBD-ACE2 interface were visualized in UCSF ChimeraX and hydrogen-bond-forming residues were shown. The BtCoV/16227–9/P.aur/DK/2020 spike (orange) and P. abramus ACE2 (green) is depicted in (A) and modelling and visualizations were done for the HKU5 RBD-P. abramus ACE2 template (PDB ID: 9D32) (B) and homology models depicting the binding interface between MERS-related RBD and ACE2 orthologs of P. auritus (C), P. kuhlii (D), E. fuscus (E) and H. sapiens (F). Homology models of BtCoV/16227–9/P.aur/DK/2020 spike (orange) and DPP4 (blue) are depicted in (G) and the binding interface between RBD and DPP4 is depicted for H. sapiens DPP4 and MERS-CoV (H) and BtCoV/16227–9/P.aur/DK/2020 (I)