2.1 Cells and viruses
Duck embryonic fibroblasts (DEFs) were derived from 9-day-old duck embryos and stored in Eagle's Minimum Essential Medium (MEM) (Gibco, Shanghai, China) supplemented with 10% Newborn Bovine Serum (NBS) (Gibco, MD, USA). HEK293T cells are maintained in our laboratory and preserved in 1640 Medium (Gibco, Shanghai, China) containing 10% Fetal Bovine Serum (FBS) (Gibco, MD, USA). All cells were cultured under conditions of 37°C and 5% CO2. The parental CHv strain of DPV (GenBank login number: JQ647509) was provided by the Institute of Immunology, College of Veterinary Medicine, Sichuan Agricultural University.
2.2 Plasmid
Based on the eukaryotic vector pCAGGS, recombinant plasmids were designed, including pCAGGS-gI-Flag, pCAGGS-gI-N69-Flag, pCAGGS-gI-N265-Flag, and pCAGGS-gI-N69/78/265A-Flag (pCAGGS-gI-NQA-Flag), for the expression of recombinant gI. In short, the gI gene fragment was amplified from the DPV CHv strain, introduce a Flag tag at its C-terminus, and it was cloned into the EcoR I and Bgl II restriction enzyme-linearized pCAGGS vector. Using the oligonucleotide primers shown in Table 1, different mutations were introduced into the fragment using standard overlap PCR.
Moreover, we have introduced the "Kan" fragment into the aforementioned four recombinant vectors for the construction of infectious clones harboring gI mutations. Taking pCAGGS-gI-N69A-Flag as an example, we utilized oligonucleotide primers listed in Table 1 to amplify the "Kan" fragment from the pEP-Kan-S vector. Subsequently, this fragment was cloned into the Bgl II restriction enzyme-linearized pCAGGS-gI-N69A-Flag vector, resulting in the acquisition of the pCAGGS-gI-N69A-Flag-Kan recombinant plasmid.
2.3 Construction and rescue of recombinant viruses
The deletion infectious clones of the DPV CHv strain, DPV CHv△gI-GS1783 and DPV CHv△gI+△gE-GS1783, were provided by our laboratory and were utilized to generate the following gI mutant infectious clones: DPV CHv-gI-N69A-GS1783, DPV CHv-gI-N78A-GS1783, DPV CHv-gI-N265A-GS1783, DPV CHv-gI-NQA-GS1783, and DPV CHv△gE-gI-NQA-GS1783, respectively. Amplify linear PCR products from gI recombinant bacteria carrying Kan, and then obtain gI mutant infectious clones based on two unlabeled Red recombinations. The specific steps are based on the construction of gene restoration strains of gene-deletion viruses, utilizing the oligonucleotide primers listed in Table 1.
In order to rescue the recombinant virus, plasmids were extracted from the gI mutant infectious clones and transfected into DEFs cultured in 12-well plates. The supernatant of the transfected cells was collected upon the appearance of visible fluorescence (EGFP) and cytopathic effects (CPE), and then passage once to produce the viral stock. The recombinant viruses were all identified through sequencing. The correctly identified recombinant viruses were designated as DPV CHv-gI-N69A, DPV CHv-gI-N78A, DPV CHv-gI-N265A, DPV CHv-gI-NQA, and DPV CHv∆gE-gI-NQA, respectively.
2.4 Viral plaque assay
Infect DEFs on 6-well plates with DPV CHv or recombinant viruses at a multiplicity of infection (MOI) of 0.0001. After a 2-hour adsorption period, the inoculum virus solution was removed, and any residual virus on the cell surface was washed away. The DEFs were then overlayed with MEM containing 1% FBS and 2% methylcellulose (Solarbio, Beijing, China). Five days post-infection, the MEM containing methylcellulose was removed, and the cells were fixed with 4% paraformaldehyde for 20 minutes. Subsequently, the cells were stained with 0.5% crystal violet for 5 minutes. After several washes with PBS, visible plaques were photographed.
2.5 Western blot analysis of recombinant gI
The plasmids pCAGGS-gI-Flag, pCAGGS-gI-N69A-Flag, pCAGGS-gI-N78A-Flag, pCAGGS-gI-N265A-Flag, and pCAGGS-gI-NQA-Flag were transfected into DEFs growing in 12-well plates at a quantity of 1000 ng per well. 36 hours after transfection, cells were lysed using RIPA buffer (Beyotime, China, P0013D). The lysates were then centrifuged at 4°C and 12,000 rpm for 10 min to obtain a clarified supernatant. The supernatant was mixed with sample buffer and boiled at 100°C for 10 min. This mixture was then subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot. For Western blot detection, the primary antibodies used were anti-Flag monoclonal antibody (mAb) (M185-3L, MBL) and GAPDH (60004-1-lg, Proteintech). The secondary antibody employed was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1, Proteintech). Visualization was carried out using the Clarity Western ECL system (Bio-Rad, USA).
2.6 Deglycosylation of recombinant gI
Transfect 1000 ng of plasmid DNA per well, including pCAGGS-gI-Flag, pCAGGS-gI-N69A-Flag, pCAGGS-gI-N78A-Flag, pCAGGS-gI-N265A-Flag and pCAGGS-gI-NQA-Flag, into DEFs cultured in a 12-well plate. At a MOI of 0.01, the recombinant viruses DPV CHv, DPV CHv-gI-N69A, DPV CHv-gI-N78A, DPV CHv-gI-N265A, and DPV CHv-gI-NQA were individually used to infect DEFs cultured in 12-well plates. Following transfection or infection for 36 hours, cells were lysed using RIPA buffer. The lysates were then centrifuged at 4°C at 12,000 rpm for 10 min to obtain a clarified supernatant. Subsequently, the supernatant was boiled at 100°C for 10 min, and Peptide N-glycosidase F (PNGase F) was added according to the manufacturer's instructions (P0704S, NEB). The sample was then incubated at 37°C for 1 hour to allow for enzymatic deglycosylation. Then, the reactants were subjected to 12% SDS-PAGE, followed by the Western blot analysis as described above. The detection of recombinant virus gI was performed using a mouse polyclonal antibody against DPV gI, prepared in this laboratory, as the primary antibody. A rabbit polyclonal antibody against DPV VP5 served as a control for viral infection, and GAPDH was used as a loading control.
2.7 Subcellular localization of recombinant gI
For co-localization analysis, DEFs were plated onto glass slides in 12-well plates. The following transfections or infections were conducted: (1) DEFs were co-transfected with 500 ng of various plasmids pCAGGS-gI-Flag, pCAGGS-gI-N69A-Flag, pCAGGS-gI-N78A-Flag, pCAGGS-gI-N265A-Flag, pCAGGS-gI-NQA-Flag, and 500 ng of pDsRed2-ER. (2) DEFs were co-transfected with 500 ng of pCAGGS-gI-Flag, pCAGGS-gI-NQA-Flag, and 500 ng of pCAGGS. (3) DEFs were co-transfected with 500 ng of pCAGGS-gI-NQA-Flag and 500 ng of pCAGGS-gE-myc. (4) DEFs were infected with 0.01 MOI of DPV CHv△gE-gI-NQA and DPV CHv-gI-NQA. After a 36-hour incubation period, indirect immunofluorescence assay (IFA) was conducted as previously described[35]. The cells were incubated with the specified primary antibody (diluted 1:200) overnight at 4°C, followed by a 2-hour incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (F-2761, Invitrogen)/Alexa Fluor 586-conjugated goat anti-rabbit IgG (T-2769, Invitrogen) as the secondary antibodies (diluted 1:1000). The cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) (D3571, Invitrogen) at room temperature for 5 min. Finally, the glass slides were examined under a fluorescence microscope.
2.8 Co-immunoprecipitation
Transfect 1000 ng of pCAGGS-gI-Flag, pCAGGS-gI-N69A-Flag, pCAGGS-gI-N78A-Flag, pCAGGS-gI-N265A-Flag and pCAGGS-gI-NQA-Flag along with 1000 ng of pCAGGS-gE-myc into HEK 293T cells. Following transfection for 36 hours, cells were lysed using RIPA buffer. The lysate was then centrifuged at 4°C at 12,000 rpm for 10 min to obtain a clarified supernatant. After centrifugation to clarify the lysate, 0.5 mL of the cell lysate from each sample was taken and incubated with 10 μg of the indicated antibody and 1 mg of SureBeads Protein G (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 2 hours. The SureBeads Protein G were washed 3 times with PBST (phosphate buffer solution) and centrifuged at 13,000 ×g for 1 min. Ultimately, the precipitates were resolved by 12% SDS-PAGE, and Western blot was performed using appropriate antibodies. Visualization of the blots was achieved using the Clarity Western ECL system.
2.9 In vivo experiments
Ducks at 14 days of age were inoculated with 107 TCID50 DPV CHv (n=10), DPV CHv-gI-NQA (n=10), and MEM (n=10), respectively. The clinical signs of the ducks in each group were continuously monitored, including percent survival, rectal temperature, and body weight. Subsequently, 14-day-old ducks were re-inoculated with 107 TCID50 DPV CHv (n=10), DPV CHv-gI-NQA (n=10), and MEM (n=10), respectively. Infected ducks were regularly necropsied to observe the histopathological changes in different organs. At the end of the experiment, all surviving ducks were humanely euthanized.
2.10 Quantification of DPV gene copy number
Quantification of DPV genome copy number in duck viscera. In each group of infected animals, heart, liver, spleen, glandular stomach, duodenum, cecum, bursa of Fabricius and thymus samples were collected on the 5th and 8th day after infection, and virus replication in vivo was monitored by quantification of DPV gene copy number. DNA was isolated from all samples using a cell and tissue genomic DNA extraction kit according to the manufacturer's instructions (Magen; Catalog number: D3191-03). As mentioned earlier, the DPV genome copy number was detected by qPCR[35].
2.11 Data analysis
All experiments were conducted in triplicate and statistical analysis was performed using GraphPad Prism 9. One-way analysis of variance (ANOVA) was used for virus plaque size data. The survival curve of ducks was analyzed by logarithmic rank (Mantel-Cox) test. t-test was used to analyze the viral load of recombinant virus in different tissues. The P value <0.05 was considered statistically significant.
2.12 Ethics statement
All 1-day-old ducks were purchased from a farm operated by Sichuan Agricultural University (Sichuan, China). All ducks were DPV free and DPV antibody negative. All ducks are housed in animal facilities at Sichuan Agricultural University in Chengdu, China. This study was approved by the Experimental Operation Guidelines and Animal Welfare Committee of Sichuan Agricultural University (Approval license No. XF2014-18).