Circulation of SARS-CoV–Related Coronaviruses and Alphacoronaviruses in Bats from Croatia

Bats are natural hosts of various coronaviruses (CoVs), including human CoVs, via an assumed direct zoonotic spillover or intermediate animal host. The present study aimed to investigate the circulation of CoVs in a bat colony in the Mediterranean region of Croatia. Guano and individual droppings from four bat species were sampled and tested with the E-gene sarbecovirus RT-qPCR, the pan-CoV semi-nested RT-PCR targeting the RdRp gene and NGS. Furthermore, bat blood samples were investigated for the presence of sarbecovirus-specific antibodies with the surrogate virus neutralization test (sVNT). The initial testing showed E-gene Sarebeco RT-qPCR reactivity in 26% of guano samples while the bat droppings tested negative. The application of RdRp semi-nested RT-PCR and NGS revealed the circulation of bat alpha- and betaCoVs. Phylogenetic analysis confirmed the clustering of betaCoV sequence with SARS-CoV–related bat sarbecoviruses and alpha-CoV sequences with representatives of the Minunacovirus subgenus. The results of sVNT show that 29% of bat sera originated from all four species that tested positive. Our results are the first evidence of the circulation of SARS-CoV–related coronaviruses in bats from Croatia.