The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-ΔUS3 and BAC-CHv-ΔUS3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-tocell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.Duck plague, also known as duck viral enteritis (DVE), is an acute, febrile, septic disease that appears in waterfowl and is caused by duck plague virus (DPV). Duck plague spreads rapidly with high morbidity and mortality, resulting in significant economic losses in the avian industry. DPV belongs to the Alphaherpesvirinae subfamily and contains a double-stranded helical DNA consisting of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region, thus forming the UL-IRS-US-TRS structure of the viral genome 1-3 .The characteristics of some genes of DPV have been reported. Herpesvirus genes are classified into immediate-early (IE), early (E) and late (L) genes according to their order of gene expression and thereby play different roles in viral replication. The kinetic classes of many DPV genes have been identified. DPV UL54, an IE gene, can shuttle between the nucleus and cytoplasm to regulate viral replication, and the recombinant UL54-deleted virus produced smaller viral plaque sizes and lower viral genome copies than the parental virus 4-7 . DPV UL13 is an E gene and localizes to the nucleus and cytoplasm, and knocking out UL13 impaired viral replication 8 . Most DPV genes, including US2 9 , US5 10 , US10 11 , UL16 12 , UL35 13 , UL41 14 , UL53 15 , and UL55 16 , are classified as L genes. To date, only a few proteins encoded by DPV apart from UL54 and UL13 have been studied in mutant viruses. Both ...