Combined NGS and proteomics improves viral diagnostics in wildlife: coronaviruses confirmed in hedgehogs

Introduction Metaviromic sequencing is prone to artifacts, necessitating orthogonal validation. This study evaluates a targeted proteogenomic approach for verifying viral presence initially detected by NGS. Methods We analyzed oropharyngeal/anal swabs and feces from hedgehogs, where NGS indicated viruses from the Coronaviridae , Picornaviridae , Flaviviridae , and Astroviridae families. For verification, mass spectrometry was applied using sample-specific databases derived from NGS-assembled contigs. Results Proteomic analysis confirmed Coronaviridae presence in NGS-positive animals, primarily detecting structural virion proteins in anal and fecal samples. However, proteomic evidence for Picornaviridae , Flaviviridae , and Astroviridae was not obtained despite their genomic detection. Discussion Phylogenetic analysis of two complete Picornaviridae genomes assembled from the NGS data – one closely related to a UK rodent virus and another distantly related to Chinese bat viruses – supports the authenticity of the NGS findings. The results demonstrate that this two-stage pipeline provides critical orthogonal validation for metaviromic studies but also defines a practical sensitivity limit for the proteomic verification of some viral families. Combining NGS with proteomics significantly enhances the reliability of viral confirmation in complex samples, thereby strengthening ecological and zoonotic inferences.