Avian herpesvirus-specific LORF5 is a late gene, interacts with 19 viral and 111 host proteins, critical for virulence of Duck plague virus.

Duck plague virus (DPV) poses a major threat to the waterfowl industry. Its genome encodes multiple avian herpesvirus-specific genes with unknown functions, which hinders the understanding of the mechanism underlying DPV pathogenesis. LORF5 is among the more conserved genes, yet the properties and biological roles of its encoded product remain unclear. Here, we systematically characterized the basic biological features of DPV LORF5 and investigated its contribution to viral replication and virulence. Bioinformatic analyses, Western blotting, indirect immunofluorescence, and pharmacological inhibition assays were used to evaluate gene expression kinetics and protein characteristics. In addition, a LORF5 deletion mutant (DPV-ΔLORF5) and the corresponding revertant were generated using bacterial artificial chromosome (BAC) technology, and virulence was evaluated in a duckling model. The host interactome of LORF5 was further profiled by coimmunoprecipitation coupled with LC-MS/MS. We demonstrate that LORF5 encodes an approximately 100-kDa protein and is expressed as a late gene. The LORF5 protein exhibited dynamic subcellular localization, residing predominantly in the cytoplasm early after infection and progressively translocating to the nucleus at later stages. In vivo, compared with the parental and revertant viruses, DPV-ΔLORF5 displayed markedly reduced replication in ducks (approximately 100-fold) along with complete loss of lethality and pathogenicity, with inoculated ducklings showing no clinical signs or pathological lesions typical of DPV infection. Interactome analysis revealed extensive interactions between LORF5 and host proteins involved in the cytoskeleton (actin/myosin) and metabolic pathways, suggesting the potential roles of LORF5 in intracellular transport and metabolic reprogramming. Collectively, these findings identify LORF5 as a novel DPV late protein and demonstrate that it is a critical virulence determinant.