Complete Genome Sequence and Construction of an Infectious Bacterial Artificial Chromosome Clone of a Virulent Duck Enteritis Virus Strain XJ

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.