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The Us4 gene homologue of duck enteritis virus (DEV) was cloned by degenerate polymerase chain reaction (PCR) and sequenced. DEV Us4 gene open reading frame (ORF) was 1380 bp in length and its primary translation product was a polypeptide of 459 amino acids long. Phylogenetic tree of the nucleotide sequences showed this gene had a close evolutionary relationship with GaHV-1 and PsHV-1, but the DEV CHv formed a solitary branch, indicating that the DEV should be placed into a single cluster within the subfamily Alphaherpesvirinae. In the analysis of rare condons, there were 36 rare condons and 4 consecutive rare codons (AGGATA, ATACGA, CTACTA, CTAAGA) in the ORF of the DEV-Us4 gene. Through combining the results of estimating secondary structure and predicted epitopes, nine B cell dominant epitopes could be initially definited, which located at 21-29, 56-74, 128-139, 182-189 194-202, 228-241, 286-299, 314-325, and 361-383 amino acids. In conclusion, all data will contribute to us understanding of this virus at the molecular level and provide a prerequisite for further functional analysis of this gene.