Cells and viruses
The DEV strain CHv (GenBank No. JQ647509.1) was procured from Avian Disease Research Center of Sichuan Agricultural University. Duck embryo fibroblast (DEF) cells were maintained in modified Eagle’s Minimum Essential Medium (MEM) (Thermo Fisher, USA) supplemented with 10% bovine serum at 37 °C in a 5% CO2 atmosphere. Human embryonic kidney (HEK) 293 T cells were maintained in Dulbecco’s Modified Eagle’s Minimum Essential Medium (Thermo Fisher, USA) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, USA), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 atmosphere.
Antibodies and vectors
The rabbit anti-UL21 polyclonal antibodies were generated for this study, and the rat anti-UL16 polyclonal antibodies were provided by He Qin . The following monoclonal antibodies were used in this study: rabbit anti-GRP78 BiP (Abcam, UK), mouse anti-TGN46 (Abcam, UK), goat anti-rabbit IgG (Thermo Fisher Scientific, USA), rabbit anti-Myc tag (Beyotime, CHN), mouse anti-Flag tag (Transgen Biotech, CHN), Alexa Fluor 594 Goat anti-Rabbit IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-rat IgG (Abcam, UK), Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies, USA), and mouse anti-β-actin (Beyotime, CHN). Normal rabbit IgG was obtained from Beyotime, and normal rat IgG was obtained from Thermo. The pCAGGS , pCMV-Myc , and pET-32c plasmids were provided by the Sichuan Agricultural University Avian Diseases Research Center.
Preparation and identification of polyclonal antibodies
pUL21 was expressed and purified via gel and electric elution. Approximately 1 mg of UL21 was emulsified in complete Freund’s adjuvant (Sigma, GER) and used to immunize rabbits through intradermal injections. Subsequent booster doses of 1 mg, 1.5 mg and 0.5 mg were prepared in incomplete Freund’s adjuvant, and the protein was administered after 2 and 3 weeks by subcutaneous injection. To collect the antibodies, the rabbits were bled through an ear vein 1 week after the final immunization. The antiserum was harvested, and preliminary purification was conducted using saturated ammonium sulfate. The antibody production followed the Sigma polyclonal antibody production method.
For the western blotting (WB), lysates were separated by SDS-PAGE, and then, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA), which was subsequently blocked with blocking buffer (5% skim milk and 0.1% Tween 20 in PBS) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with rat anti-UL16 or rabbit anti-UL21 monoclonal antibodies at dilutions of 1:100 or rabbit anti-Myc or mouse anti-Flag polyclonal antibodies at dilutions of 1:1000. Then, the membrane was washed three times with PBST and incubated with HRP-conjugated goat anti-rabbit IgG, goat anti-mouse IgG or goat anti-rat IgG (1:3000) secondary antibodies for 1 h at 37 °C. The membrane was washed three times with PBST, and the signals were developed using an enhanced chemiluminescence (ECL) kit (Takara, JPN).
Quantitative reverse transcription PCR
The total RNA was isolated from the DEV-infected DEFs at different time points (3, 6, 13, 17, 24, 36, 48 and 60 hpi), and then, reverse transcription was performed; an uninfected control was included. The primers were designed with Oligo 7 (Additional file 1: Table S1). Quantitative reverse transcription PCR was performed in a 20-μL reaction volume containing 10 μL of SYBR Green mix (Takara, JPN), 1 μL of each primer, 1 μL of cDNA, and 7 μL of RNase-free water. Triplicate experiments were performed to analyse UL21, UL54, UL13, US2 and β-actin gene expression, and the relative transcription levels were calculated using the 2-ΔCt method .
Pharmacological inhibition was performed to confirm the DEV UL21 gene expression patterns. Three flasks of DEFs were prepared and inoculated with DEV as follows: one flask was prepared without any drugs, and the other flasks contained either 300 μg/mL ganciclovir (GCV, a DNA polymerase synthesis inhibitor) or 100 μg/mL cycloheximide (CHX, a protein synthesis inhibitor). The total RNA was isolated from the DEV-infected DEFs incubated with GCV or CHX (Meilunbio, CHN) at 24 hpi and subsequently reverse-transcribed into cDNA. Then, the cDNA was used for a PCR analysis.
Cells grown on coverslips were washed three times with PBS and fixed overnight with 4% paraformaldehyde in PBS at 4 °C. For the indirect immunofluorescence analysis (IFA), the fixed cells were permeabilized with 1% Triton X-100 in PBS for 30 min at 4 °C and incubated with 200 μL of blocking buffer (3% bovine serum albumin in PBS) in a humidified chamber for 1 h at 37 °C. Then, the cells were incubated with primary antibodies (rabbit anti-UL21 and rat anti-UL16 at a dilution of 1:200) and Alexa Fluor-conjugated secondary antibodies (at a dilution of 1:1000) in blocking buffer for 60 min at 37 °C. The samples were examined under a Nikon H550L fluorescence microscope.
The cells were transfected at 90 to 95% confluence with 2.5 μg of plasmid DNA added to 125 μL of MEM and mixed well; then, 3.75 μL of Lipofectamine 3000 (Thermo Fisher Scientific, USA) in 125 μL of MEM were added, and the cells were gently mixed and incubated at room temperature for 5 min. The DNA suspension and 4 μL of p3000 were mixed together and incubated at room temperature for 15 min, and then, the mixture was added to a 6-well plate. The plate was shaken gently and placed in a 37 °C cell incubator.
The DEFs were infected with the DEV strain CHv at a multiplicity of infection (MOI) of 0.2. The infected DEFs were washed twice with cold PBST, and PMSF was added to the immunoprecipitation (IP) cell lysis buffer (Beyotime, CHN) at a final concentration of 1 mM. Precooled IP cell lysis buffer at 100 μL/mL was added to the cells, which were scraped from the plates, placed on ice, and shaken slowly on a horizontal shaker for 15 min until they were fully lysed. The cells were centrifuged at 14,000×g for 15 min at 4 °C, and the supernatant was collected. Protein A + G agarose (Bio-Rad, USA) was washed three times with PBST. Rat anti-UL16 IgG and rabbit anti-UL21 IgG (rat anti-UL16 and rabbit anti-UL21 monoclonal antibodies at dilutions of 1:10) were added to the agarose beads. Rabbit anti-Myc or mouse anti-Flag polyclonal antibodies were also used at dilutions of 1:100. The samples were gently rotated at room temperature for 30 min. Then, the complexes were washed three times with PBST. The lysates containing the target proteins were added, and the mixture was incubated at 4 °C overnight under gentle rotation. The samples were washed with PBST, the complexes were rapidly centrifuged for 30 s, and the supernatants were collected. Finally, 1 × SDS loading buffer was added, and the samples were heated for 10 min at 70 °C.
SDS-PAGE was used to separate the purified virion samples. The products were stained with Coomassie brilliant blue (Bio-Rad, USA) and then sent to Sangon Biotech Company (Sangon Biotech, CHN) for a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The methods used for the in-gel trypsin digestion, LC-MS/MS, and database searches have been described in detail by Loret et al. .
The DEFs were infected with the DEV strain CHv at an MOI of 5. At 2 hpi, the cells were washed twice with PBS, and the medium was replaced with Opti-MEM. At 72 hpi, the medium was collected and clarified by centrifugation at 2000×g for 20 min at 4 °C to remove the cell debris. The DEV virions were harvested by ultracentrifugation (40,000×g, 2 h, 4 °C) through a 30% (wt/vol) sucrose cushion and then banded by isopycnic gradient ultracentrifugation in a continuous 30 to 60% (wt/vol) potassium tartrate gradient in TBS (40,000×g, 2 h, 4 °C). The band containing the virions was collected, diluted tenfold in TBS, and pelleted by ultracentrifugation (20,000×g, 30 min, 4 °C). The pellets were resuspended in TBS and stored at − 80 °C .