Duck embryo fibroblasts (DEFs) were cultured in minimum essential medium (MEM; Thermo Fisher Scientific, United States) supplemented with 10% newborn calf serum (NBS) at 37°C in a 5% CO₂ incubator. Maintenance medium supplemented with 2% NBS was used for the viral infection. DEV strain CHv (GenBank: JQ647509.1) was isolated, preserved, and supplied by our laboratory.

The E. coli strain DH5α; E.coli BL21pLysS, pMD20-T, pET-32a (+), and pCAGGS vectors; and pCAGGS–UL51–Flag plasmid were provided by our laboratory. A rabbit anti-UL7 polyclonal antibody was generated for this study; rabbit anti-DEV and anti-UL51 polyclonal antibodies were prepared as described earlier (Shen et al., 2009). HRP-conjugated goat anti-rabbit IgG (Life Technologies, United States), mouse anti-β-actin (Beyotime, CHN), FITC-conjugated anti-rabbit IgG (Abcam, UK), anti-HA-tagged rabbit (Beyotime, CHN), anti-Flag-tagged mouse (Transgen Biotech, CHN), Alexa Fluor 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, United States), and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Life Technologies, United States) were used in this study.

The primers were designed with Oligo 7.0 and primer 5.0 (Table 1). The UL7 gene (GenBank: FJ222445) of the DEV strain CHv was inserted into the pMD20-T clone vector, digested with BamHI and XhoI, and ligated into E. coli expression vector pET-32a (+) (Invitrogen) to create the plasmid pET32a–UL7. The plasmids expressing UL7–HA and UL51–Flag from DEV were constructed by PCR amplification of the coding region and subcloned into the pCAGGS vector to create pCAGGS–HA–UL7 and pCAGGS–UL51–Flag, respectively, and transfected into DEFs using the HieffTrans™ liposomal transfection reagent (Yeasen, CHN) according to the manufacturer’s protocol.

The recombinant pUL7 of DEV was purified under denaturing conditions because it was expressed in E. coli BL21pLysS with inclusion bodies. To obtain the highest pUL7 expression, the conditions were optimized by inducing different IPTG concentrations, temperatures, and time periods. All expression levels were detected by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (SDS–PAGE).

The DEV pUL7 was purified by gel and electric elution and used to prepare the polyclonal antiserum of pUL7. Briefly, four male New Zealand white rabbits (Chengdu dossy experimental animals CO., Ltd.) were immunized intradermally with 0.5 mg pUL7 emulsified in Freund’s complete adjuvant. Subsequently, Freund’s incomplete adjuvant and 1.0 mg pUL7 were injected. Two boosted subcutaneous injections were administered at 2 weeks interval after the primary injection. One week later, the rabbits were intravenously injected with 0.1 mg purified pUL7. Two weeks after the last immunization, the rabbits were bled and antiserum was prepared. Control preimmune serum samples were obtained from non-vaccinated healthy rabbits. The rabbit polyclonal antiserum was purified using ammonium sulfate precipitation (McGuire et al., 1996). In addition, the antibody titer of the pUL7 antiserum was confirmed using a double-diffusion assay in 1% agarose.

Protein samples were lysed in SDS sample buffer, separated by SDS–PAGE, and immediately transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, United States). Nonspecific protein binding was blocked by treating the membranes with PBS containing 5% skim milk at room temperature for 1 h. The membranes were washed with PBST (PBS containing 0.05% Tween 20) and incubated overnight at 4°C with appropriately diluted primary antibodies. After washing three times with PBST, the membrane was incubated with the HRP-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies at 37°C for 1 h. The membranes were then washed three times with PBST, treated with an enhanced chemiluminescence (ECL) kit, and exposed on a BioRad CheminDOC XRS+ system.

Total RNA was isolated from DEV-infected cells at 0, 1, 2, 4, 8, 12, 16, 24, 32, 40, 48, 56, and 64 h post-infection using a Total RNA Isolation System (Takara, JPN). The purified RNA was immediately inverse transcribed to cDNA by PrimeScript^® RT reagent kit with gDNA Eraser (Takara) according to the manufacturer’s instructions. The primers were designed by Oligo 7 (Table 1), and RT-qPCR was performed in 20 μL reaction volumes containing 1 μL each primer, 1 μL cDNA, 10 μL SYBR Green Mix, and 7 μL RNase-free water in triplicate. β-actin was used as an internal control. Average relative content of DEV UL7 gene transcription was calculated by the 2^−ΔΔCT method (Liu et al., 2015).

To examine the DEV UL7 gene expression patterns, DEFs were infected with the DEV strain CHv in three T25 flasks; one flask was treated with 300 μg/mL acyclovir (ACV), the second was treated with 100 μg/mL cycloheximide (CHX), while the third was prepared without any drugs as control. Total RNA was extracted from DEV-infected DEFs and the control group at 24 h post-infection (hpi) and subsequently reverse-transcribed into cDNA. PCR was performed using the primers for UL54, UL13, US2, UL7, and β-actin genes (Table 1).

The cells were fixed overnight in 4% paraformaldehyde at 4°C after washing three times with PBS. The cells were then permeabilized with 0.2% Triton X-100 for 10 min and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 37°C. The cells were labeled with primary antibodies at 4°C overnight and Alexa Fluor-conjugated secondary antibodies in blocking buffer for 1 h at 37°C. After that, the cells were stained with 4′6′-diamidino-2-phenylindole (DAPI) for 5 min after washing three times again at room temperature. Finally, the fluorescent images were examined using a Nikon H550L imaging system.

Proteins were immunoprecipitated from DEFs transfected with the pCAGGS–HA–UL7 and pCAGGS–UL51–Flag plasmids for 24 h. Transfected DEF monolayers of 50 mm cultures were washed twice with 3 mL cold PBST, and the cells were suspended in 1 mL cold PBST and pelleted at 110 × g for 10 min. The cell pellets were resuspended in 0.4 mL IP buffer (Yeasen, CHN) supplemented with PMSF (final concentration: 1 mM), transferred to new microcentrifuge tubes, and placed on ice for 5 min. Then, the cells were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was transferred to a new tube. After removal of a fraction of the sample as a control, 2 µL mouse anti-Flag and rabbit anti-HA IgG were added and the mixture was incubated at 4°C overnight under gentle rotation. On the next day, 20 µL protein A agarose (Bio-Rad, United States) was added and the samples were incubated and gently rotated at 4°C for 3 h. The samples were then washed thrice with PBST, rapidly centrifuged for 30 s, and the supernatant was collected. Finally, the samples were heated for 10 min at 90°C with 5× SDS loading buffer.

The recombinant plasmid pET32a–UL7 was transformed into E. coli BL21pLysS and expressed mainly in the insoluble fraction (50 kDa). pUL7 was purified and analyzed using SDS–PAGE (Figure 1A). The immunogenicity of the purified UL7 fusion protein was detected using rabbit anti-DEV antibodies, which specifically recognize a 50 kDa molecule (Figure 1B). Rabbit anti-pUL7 polyclonal antibodies (titer: 1:32) were generated in this study, which can specifically recognize pUL7 in DEV-infected DEFs (molecular weight: approximately 35 kDa), which is consistent with the expected size of DEV pUL7 (Figure 2D).

The amplification efficiency of the target and reference gene must be approximately equal for applying the 2^−ΔΔCT method. Both standard curves showed the same PCR efficiency (Figure 2A) and their melting curves presented a single peak (Figure 2B). The UL7 transcripts in DEV-infected cells were analyzed by real-time quantitative PCR (RT-qPCR) with SYBR Green I. The average relative DEV UL7 gene transcript content was calculated by the 2^−ΔΔCT method. The results indicated that DEV UL7 gene transcripts were not detected in negative controls (at 0 hpi), first detected at 4 hpi, increased steadily until reaching a peak at 56 hpi, and maintained a high level at 64 hpi with a descending trend (Figure 2C).

The cell lysates collected at different time points after DEV post-infection were analyzed by western blotting, and the PVDF membranes were probed with a rabbit anti-pUL7 polyclonal antibody (Figure 2D). Band density was analyzed using ImageJ software, which showed that pUL7 expression began at 12 hpi and peaked 36 hpi. As a control, the same cell lysates were probed with a monoclonal antibody against β-actin, and the β-actin levels remained relatively constant.

Previous studies have shown that many DEV genes are late genes after acyclovir (ACV) or cycloheximide (CHX) treatment (He et al., 2018; Yang et al., 2020). Therefore, we examined the effects of ACV and CHX on DEV UL7. Figure 3 illustrates the correct bands of the immediate-early (IE) gene UL54, early (E) gene UL13, and late (L) gene US2; β-actin was detected as a control and the negative group was DEFs untreated with ACV or CHX. UL7 showed the same band as US2 and was observed only in the DEV-infected group without ACV or CHX treatment. These results indicated that UL7 is an L gene in DEV.

The intracellular localization of pUL7 in DEV-infected DEFs was examined by indirect immunofluorescence assays using pUL7 antiserum. The pUL7-specific fluorescent was detected in the cytoplasm at 8 hpi (Figure 4). At 48 hpi, the pUL7-specific fluorescent granules had increased and clustered strongly in the perinuclear region. However, no specific staining was observed in mock-infected DEFs and infected DEFs at 4 hpi. Taken together, these observations indicate that DEV pUL7 is predominantly localized in the cytoplasm of DEV-infected DEFs.

To determine whether DEV pUL7 co-localization with pUL51, we first created pCAGGS–HA–UL7 and pCAGGS–UL51–Flag vectors and expressed them via transfection into DEFs individually or together. When cells were infected alone, pUL7 was mainly observed in the cytoplasm (Figure 4). In addition, pUL51 was detected mainly in the cytoplasm (Figure 5A). When pUL7 was co-expressed with pUL51, a juxtanuclear Golgi apparatus-like distribution was observed, and merged fluorescence analysis showed that the two proteins were co-localized in the cytoplasm and nucleus (Figure 5B). However, this did not necessarily imply an interaction during co-expression. To confirm that the pUL7–pUL51 complex could form in co-transfected DEFs, we detected the complex using co-immunoprecipitation (Co-IP). The results showed that pUL7 co-immunoprecipitated with pUL51 in the co-transfected DEFs (Figure 6).