Coronavirus Diversity in an Italian Bat Population Sampled During the Pandemic in 2020

Evolutionary studies have established a correlation between human and bat coronaviruses (CoV), identifying bats as the natural reservoir for Coronaviridae. Over the past two decades, three major zoonotic spillover events have been described: Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV), which caused significant global outbreaks, and SARS-CoV-2, which triggered the COVID-19 pandemic. Despite these events, the true diversity of coronavirus species harbored by bats remains largely unmapped, even as the discovery of novel strains continues to accelerate. In this study, we analyzed 37 fecal swab samples collected in Northern Italy, targeting the bat genera Miniopterus, Myotis, and Rhinolophus. Coronaviruses were detected using a pan-coronavirus (Pan-CoV) nested RT-PCR in five bat samples: three tested positive for Alphacoronaviruses (Alpha-CoVs) and two for Betacoronaviruses (Beta-CoVs). Positive samples were subjected to Pan-CoV Illumina next-generation sequencing (NGS), and two samples yielded sufficient read depth for robust contig assembly. Phylogenetic analysis revealed the co-circulation of three distinct Alpha-CoVs within a Miniopterus schreibersii (M. schreibersii) fecal sample and the presence of a SARS-CoV-related strain in a Rhinolophus hipposideros (R. hipposideros) fecal sample. Both the Alpha-CoVs and the Beta-CoVs were most closely related to European strains. These findings confirm the widespread circulation of diverse CoVs in Italian bats and underscore the need for continued molecular epidemiological surveillance to unravel the full extent of CoV heterogeneity in European wildlife.