Functional characterization of UL50 gene reveals its essential role in duck enteritis virus replication and pathogenesis

Figure 1.. Multiple sequence alignment of UL50-encoded proteins. Comparison of the UL50-encoded protein from DEV with homologous proteins from BHV-1, HSV-1, PRV, and VZV. Sequences were aligned using Clustal Omega. Gaps in the alignment are indicated by dashes (‘-’); symbols ‘*’, ‘:’, and ‘.’ represent identical, conserved, and semi-conserved residues, respectively. Different colours denote different amino acid properties. Conserved motifs are highlighted with grey shading.

Figure 2.. Predicted structural features and Subcellular localisation of the DEV UL50-encoded protein. (A) AlphaFold2 prediction of the 3D structure of the UL50-encoded protein, with colours indicating prediction confidence (pLDDT score): blue (very high, >90), cyan (confident, 70–90), yellow (low, 50–70), and red (very low, <50). (B) Visualisation of the six conserved motifs in the predicted structure using PyMOL, with each motif highlighted in a different colour. (C) Structure superimposition of DEV UL50 protein (light blue) with homologous proteins from HSV-1, PRV, BHV-1, and VZV (violet), performed using PyMOL. The root mean square deviation (RMSD) values indicate structural similarity. (D) Nuclear localisation signal (NLS) prediction using DeepLoc. A prominent peak was observed around positions 445–450. (E) Subcellular localisation of DEV UL50 protein in DEFs. Cells were either mock-transfected or transfected with pUL50-Flag. At 24 and 48 hpi, cells were fixed and stained with anti-FLAG (DYKDDDDK) rabbit polyclonal antibody followed by Cy3-conjugated goat anti-rabbit IgG. Nuclei were counterstained with DAPI (blue).

Figure 3.. Construction of UL50 gene-deleted virus and its repaired strain. (A) Schematic representation of the two step red-mediated recombination to process used to construct the ΔUL50 strain. The UL50 genomic region and its flanking genes (UL49.5 and UL51) are shown. The entire UL50 open reading frame was deleted. (B) The BAC-free UL50-deleted strain (ΔUL50), UL50-repaired strain (RUL50), and parental strain (WT) were harvested following rescue and purification. (C) RFLP analysis of pXJ-BAC-ΔUL50 and pXJ BAC-RUL50. The left panel shows the predicted patterns generated using Vector NTI, while the right panel presents the experimental results. Red arrows indicate differences among recombinant viruses observed after BamHI digestion, while blue arrows highlight variations following XhoI digestion. (D) PCR analysis confirming the identity of recombinant viruses. (E) Western blot analysis verifying the expression of the DEV UL50 protein. Cell lysates from recombinant virus-infected cells were subjected to Western blotting and probed with anti-UL50 polyclonal antibody. β-actin served as a loading control.

Figure 4.. Comparison of biological characteristics among ΔUL50, RUL50, and WT strain. (A) Multistep growth kinetics of recombinant viruses. Viral titre in infected cells were measured at different time points following inoculation with approximately 0.02 MOI of virus. Statistical analyses were performed on the total, cell, and supernatant viral titre at each time point. Growth curves represent the mean values from three independent experiments. (B) Viral adsorption assays. DEFs were infected with the indicated viruses at 4 °C, and viral DNA copy numbers were quantified from collected samples. (C) Viral penetration assays. Following adsorption, DEFs were incubated at 37 °C for 1 hour, and viral DNA copy numbers were quantified from collected cell samples. (D) Viral replication assay. Cell samples were collected at 6, 9, and 12 hours post-infection, and viral DNA copy numbers were determined. (E) Plaque morphology of recombinant viruses. Upper panel: DEFs were infected with WT, ΔUL50, and RUL50 at the same MOI and stained with crystal violet at 6 days post-infection. Lower panel: Recombinant viruses carrying a GFP reporter gene were used to further visualise plaque formation. (F) Plaque…

Figure 5.. Pathogenesis of ΔUL50, RUL50, and WT strain. (A) Survival curve. Ducks (n = 10 per group) were infected with ΔUL50, RUL50, or WT strains, with DMEM used as a negative control. Survival rates were monitored over time. (B) Rectal temperature. The rectal temperatures of infected ducks were measured daily for 10 days post-infection, and temperature trends for each group were plotted. (C) Histopathological analysis. Autopsy findings from infected ducks. Yellow arrows indicate organ lesions. In the WT and RUL50 groups, pronounced haemorrhagic spots were observed in the oesophagus and cloaca, along with circular haemorrhagic spots on the intestinal surface. In contrast, no lesions were detected in the ΔUL50 group or the negative control group. (D) Microscopic observation of visceral lesions in ducks post-infection. Sheldrake, liver, intestines and thymus, HE staining, 1 bar = 5 μm. In the liver of the WT and RΔUL50 groups, significant blood cell accumulation, pronounced haemorrhage, and cellular degeneration and necrosis were observed. Large areas of epithelial cells from the intestinal villi were detached and released into the intestinal lumen. Thymic lobules showed indistinct…

Figure 6.. Replication characteristics of recombinant virus in vivo. Tissue samples were collected from infected ducks at indicated time points post-infection to assess viral replication by qPCR. DEV viral load were quantified in the liver (A), brain (B), Thymus (C), heart (D), spleen (E), and intestines (F) (n = 5;ns, P > 0.05;*, P < 0.05**;**, P < 0.01;***, P < 0.001).

Figure 7.. Replication of recombinant viruses in DRG neurons. (A) Viral replication assay. DRG neurons were infected with ΔUL50, RUL50, and WT at an MOI of 0.1. Samples were collected at 24, 48, 72, and 96 hpi, and viral DNA copy numbers were quantified by qPCR. (B) Fluorescence intensity analysis. DRG neurons were infected with ΔUL50 BAC, RUL50 BAC, and XJ BAC (each carrying a GFP reporter gene) at an MOI of 0.1. Fluorescence intensity was quantified by randomly selecting multiple fields of view using ImageJ. (C) Viral spread assessment. EGFP expression was used to monitor the proliferation of recombinant viruses in DRG neurons. (ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001).

Figure 8.. Replication of recombinant viruses in DEFs. (A) Viral replication assay. DEFs were infected with ΔUL50, RUL50, and WT at an MOI of 0.1, simultaneously with DRG neurons. Samples were collected at 24, 48, 72, and 96 hpi, and viral DNA copy numbers were quantified by qPCR. (B) Fluorescence intensity analysis. DEFs were infected with ΔUL50 BAC, RUL50 BAC, and XJ BAC at an MOI of 0.1, simultaneously with DRG neurons. Fluorescence intensity was analysed by randomly selecting multiple fields of view using ImageJ. (C) Viral spread assessment. EGFP expression was used to monitor the proliferation of recombinant viruses in DEFs. (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ****, P < 0.0001).