Proteogenomic analysis of Cyprinid herpesvirus 2 using high-resolution mass spectrometry

Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for the development of herpesviral hematopoietic necrosis disease (HVHND) in crucian carp (<i>Carassius auratus</i>). The CyHV-2 genome encodes approximately 150 genes that are expressed in a well-defined manner during productive infection. However, CyHV-2 open reading frames (ORFs) are primarily derived from sequence and homology analyses, and most lack protein-level evidence to support their properties. In this study, we used high-resolution mass spectrometry followed by proteogenomic mapping to achieve genome re-annotation of CyHV-2. Based on our results, a total of 1,683 MS/MS spectra could be mapped to the CyHV-2 genome through six-frame translation, with 1,665 corresponding to 117 currently annotated protein-coding ORFs. Three of the remaining 18 peptides were mapped to the N-terminal extension region of known ORFs. However, 12 novel CyHV-2 ORFs, designated nORF1-12, were identified and characterized for the first time based on the remaining 15 peptides that could be mapped to previously unannotated regions of the viral genome. And the sequence differences of the novel phosphorylated nORF1, also referred to as ORF25E, in different CyHV-2 strains indicated that the nORF1 is a prospective molecular marker that can monitor the evolution from the Japan (J) to the China (C) genotype of CyHV-2. These findings further validate existing annotations, expand the genomic landscape of CyHV-2, and provide a rich resource for aquatic virology research.IMPORTANCECyHV-2 is a viral pathogen that poses a significant threat to crucian carp farming. CyHV-2 has a large genome with complex sequence features and diverse coding mechanisms, which complicates accurate genome annotation in the absence of protein-level evidence. Here, we employed various protein extraction and separation methods to increase viral protein coverage and performed an integrated proteogenomic analysis to refine the CyHV-2 genome annotation. A total of 129 viral genes were confidently identified, including 117 currently annotated genes and 12 novel genes. For the first time, we present large-scale evidence of peptide presence and levels in the genome of aquatic viruses and confirm the majority of the predicted proteins in CyHV-2. Our findings enhance the understanding of the CyHV-2 genome structure and provide valuable insights for future studies on CyHV-2 biology.