Target enrichment metaviromics enables comprehensive surveillance of coronaviruses in environmental and animal samples.

The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.