Figure 1. Analysis of truncated-UL13 protein prokaryotic expression and UL13 expression in DEV-infected DEF cells. (A) Expression and purification of the truncated DEV UL13 fusion protein. M, standard protein marker (Bio-Rad Code: 161–0317); Lane 1, total cell lysate from bacteria carrying the recombinant plasmid pET-32c(+)-UL13T without IPTG induction; Lane 2, total cell lysate from bacteria carrying the recombinant plasmid pET-32c(+)-UL13T after IPTG induction; Lane 3, the purified truncated DEV UL13 fusion protein. The predicted molecular mass of the truncated DEV UL13 fusion protein is 44 kDa. (B) Recognition of the expressed truncated UL13 fusion protein by the anti-DEV antibody. M, pre-stained protein molecular weight marker (Thermo Code: #26616); Lane 1, result of rabbit anti-DEV antiserum identifying the truncated DEV UL13 fusion protein; Lane 2, result of rabbit pre-serum identifying the truncated DEV UL13 fusion protein. The membrane was developed using the Chemiluminescence Imaging System (ChemiScope 5300, CLINX, Shanghai, China). (C) SDS-PAGE analysis of cursorily purified anti-rabbit DEV UL13 truncated protein serum. M, standard protein marker (Bio-Rad Code: 161–0317); H and L of UL13IgG indicate the position of the heavy and light chains of the IgG proteins, respectively. (D) Analysis of UL13 protein expression at different time points in DEV-infected DEF. DEV-infected DEF were harvested at 4, 8, 12, 24, 36, 48, 72 and 96 h, and immunoblotting was used to analyze UL13 protein expression. Rabbit anti-UL13 and anti-β-actin antiserum were used as the primary antibodies, and HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody. The membrane was developed using ECL immunoblotting detection reagents. M, pre-stained protein molecular weight marker (Bio-Rad Code: 161–0373). From: The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture Poult Sci. 2017;96(8):2899-2907. doi:10.3382/ps/pex043 Poult Sci | © 2017 Poultry Science Association Inc.

Figure 2. Relative transcriptional analysis of DEV UL13 gene and the pharmacological inhibition tests. (A) Temporal transcription of DEV UL13 in DEV-infected DEF. The average values of DEV UL13 and β-actin gene transcripts at 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84 and 96 h post infection and mock infection were calculated using Microsoft Excel. (B) Relative transcription of DEV UL13 in DEV-infected DEF. The average values of DEV UL13 and β-actin transcripts were calculated at the corresponding post-infection time points using Excel, then the relative content of DEV UL13 transcripts was calculated using the 2^−ΔCt method and the graph was made using GraphPad Prism 6.0 software. Each time point was measured in triplicate and is presented as the mean and standard error. (C) The pharmacological inhibition tests using conventional PCR as a read-out. “+”, cDNA of DEV-infected DEF without inhibitor added; “GCV”, cDNA of DEV-infected DEF with the addition of GCV; “CHX”, cDNA of DEV-infected DEF with CHX added; “–”, cDNA of DEF without DEV-infected. The β-actin and UL13 on both sides represented primers used in the conventional PCR reaction. M, DNA marker (DL2000). From: The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture Poult Sci. 2017;96(8):2899-2907. doi:10.3382/ps/pex043 Poult Sci | © 2017 Poultry Science Association Inc.

Figure 3. The intracellular distribution of DEV UL13 protein in DEV-infected DEF. DEV- and mock-infected DEFs were fixed with 4% paraformaldehyde, and then IFA was performed. The rabbit anti-UL13 antiserum was used as a primary antibody, and goat anti-rabbit antibody conjugated to FITC (green) was used as the secondary antibody. Nuclei were stained with DAPI (blue). Images were recorded in the FITC channel (a2-f2) and the DAPI channel (a1-f1) using a 60× objective and then merged with the SPOT software (a-f). From: The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture Poult Sci. 2017;96(8):2899-2907. doi:10.3382/ps/pex043 Poult Sci | © 2017 Poultry Science Association Inc.

Figure 4. Construction and identification of DEV CHv-BAC-ΔUL13 and DEV CHv-BAC-ΔUL13R recombinant viruses. (A) Schematic representation of the DEV genome in the UL13 region and part of the sequence for DEV CHv-BAC-ΔUL13 and the repaired DEV CHv-BAC-ΔUL13R. (B) PCR identification of the viral genome fragment near the UL13 gene region. Total DNA was isolated from DEF infected with DEV CHv-BAC, DEV CHv-BAC-ΔUL13, and DEV CHv-BAC-ΔUL13R virus. Lane 1, PCR products of the mutant virus DEV CHv-BAC-ΔUL13 were 666 bp; Lane 2, PCR products of the revertant virus DEV CHv-BAC-ΔUL13R were 2,201 bp; Lane 3, the PCR products of the parental virus DEV- CHv-BAC were 2,117 bp. M1, DNA marker (DL2000); M2, DNA marker (DL15,000). (C) RFLP analysis. Lane 1, DEV CHv-BAC-ΔUL13R-G plasmids were digested with SalI restriction endonuclease; Lane 2, DEV CHv-BAC-ΔUL13-G plasmids were digested with SalI restriction endonuclease; Lane 3, DEV CHv-BAC-G plasmids were digested with SalI restriction endonuclease. Digestion of the deletion and revertant products caused the size of the 5,714 bp fragment of the parental virus (Lane 3) to change to 4,263 bp and 5,798 bp (Lane 2 and Lane 1), respectively. No extraneous alterations were evident in either clone. M, 1 kb plus DNA ladder. *: Different band. From: The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture Poult Sci. 2017;96(8):2899-2907. doi:10.3382/ps/pex043 Poult Sci | © 2017 Poultry Science Association Inc.

Figure 5. Analysis of the characteristics of the UL13-deletion mutant virus in cell culture. (A) Plaque assay. The upper figures represent the phenotypes of the plaques formed by DEV CHv-BAC, DEV CHv-BAC-ΔUL13, and DEV CHv-BAC-ΔUL13R virus infection; the lower figure shows the analysis of the mean areas of the viral plaques formed by the various viruses during infection. The digital images were analyzed using Image-Pro Plus software and the statistical analyses were performed using GraphPad Prism 6.0 software. **: P < 0.01. (B) Growth curve. DEF were infected with DEV CHv-BAC, DEV CHv-BAC-ΔUL13, or DEV CHv-BAC-ΔUL13R at a titer of 200 TCID₅₀ and harvested at 6, 12, 24, 48, 72, and 96 hours post infection. The curves were generated based on the titers of the different harvests, by testing for the TCID₅₀ using Excel. And the significant difference analyses between DEV CHv-BAC and DEV CHv-BAC-ΔUL13 were performed using GraphPad Prism 6.0 software. *: P < 0.05, **: P < 0.01. From: The duck enteritis virus early protein, UL13, found in both nucleus and cytoplasm, influences viral replication in cell culture Poult Sci. 2017;96(8):2899-2907. doi:10.3382/ps/pex043 Poult Sci | © 2017 Poultry Science Association Inc.