Coronavirus diversity and SARS-CoV-2 exposure at the wildlife-human interface in Northern Italy.

Members of the Coronaviridae family infect humans as well as domestic and wild animals. Over the past three decades, three members of this family, all with zoonotic origins, have caused significant epidemics or pandemics (SARS, MERS, and COVID-19). Despite the spread of SARS-CoV-2 being primarily driven by human-to-human transmission, various animal species are susceptible to infection and may contribute to viral circulation. Aim of this work was to monitor coronavirus (CoV) infections in wild mammals in the Emilia-Romagna region (RER), Italy, using a combined approach of molecular screening for viral RNA detection and serological testing for anti-SARS-CoV-2 antibodies. Respiratory and gastrointestinal tissue samples were collected from wild animal carcasses between 2022 and 2024. Samples were tested for SARS-CoV-2 using two RT-qPCR assays targeting the E and N genes, and for other CoVs using a nested pan-coronavirus RT-PCR followed by Sanger sequencing of positive samples. Additionally, serum samples obtained from blood, cardiac clot, or thoracic exudate were screened for antibodies against the SARS-CoV-2 nucleocapsid (N) protein, with positive samples subsequently confirmed by an ELISA targeting antibodies to the receptor-binding domain (RBD) of the Spike (S) protein, focused on variants circulating during the study period. Molecular analyses were performed on 2,238 animals, all of which tested negative for SARS-CoV-2, while 90 (79% hedgehogs) tested positive for CoVs. Among these, most sequences were consistent with coronaviruses typically reported in the respective host species. However, some exceptions - such as Betacoronavirus erinacei in fox, porcupine, hare, and roe deer, and EmbeCoV-related sequences in a porcupine - warrant further attention. Suitable serum samples were available from 1,751 animals. Overall, 65 animals tested positive for anti-N antibodies, 31 of which (22 foxes, 4 badgers, 2 hedgehogs, 1 roe deer, 1 wolf, 1 rat) were subsequently confirmed by an anti-RBD ELISA. This study provides an overview of CoVs circulation among wild mammals in RER, supporting the role of hedgehogs as reservoirs and identifying some species with evidence of exposure to SARS-CoV-2. Certain unexpected findings highlight the need for further investigations to clarify the potential for cross-species transmission.