Detection of merbecoviruses in Danish bats using a novel broad-range RT-qPCR assay
Bat-associated merbecoviruses comprise a genetically diverse group of coronaviruses with variable receptor usage and host range, highlighting the need for efficient tools for their surveillance. Here, we developed and optimized a broad-range reverse transcription quantitative PCR (RT-qPCR) assay for the detection of bat-associated merbecoviruses, including the lineage previously identified in brown long-eared bats (Plecotus auritus) in Denmark. In silico analysis predicted detection of 898 of 941 (95.4%) merbecovirus sequences, and the assay detected all bat-associated merbecoviruses included in the experimental panel. The assay was applied to 33 bats sampled through the Danish national bat surveillance program in 2025. Merbecovirus RNA was detected in two samples, one from a P. auritus and one from a Nathusius' pipistrelle (Pipistrellus nathusii), representing the first detection of a merbecovirus in P. nathusii in Denmark. Metagenomic next-generation sequencing recovered a complete merbecovirus genome from the P. nathusii sample. Phylogenetic analysis assigned the virus to the MOW clade and showed that it was closely related to the Russian MOW15-22 virus, sharing 98.67% nucleotide identity across the complete genome and 99.85% amino acid identity in the spike protein. Limited sequence data obtained from the P. auritus sample was consistent with the merbecovirus lineage previously identified in this species in Denmark. Together, these findings demonstrate the utility of broad-range RT-qPCR combined with metagenomic sequencing for surveillance and genomic characterization of bat-associated merbecoviruses.