Cells, virus and plasmids
The BSR-T7/5 (Buchholz et al. 1999) and Vero-Dog-SLAM (VDS) cells, kindly provided by the China Animal Health and Epidemiology Center, were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and containing penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL) and G418 (500 µg/mL). The wild-type CDV (wt-CDV), QN strain, was propagated in VDS cells. The QN strain-based platform of reverse genetics had been established previously in our laboratory, mainly containing four plasmids, namely one full-length cDNA clone of recombinant CDV that expressed a foreign protein, and three helper plasmids (pCAGGS-N, pCAGGS-P and pCAGGS-L).
Construction of rCDV-Gn and -Gc cDNA clones
The rCDV-Gn and -Gc cDNA clones were schematically shown in Fig. 1C and D, respectively. They were separately flanked by the T7 promoter and a hepatitis delta virus ribozyme-T7 terminator fusion sequence at their 5′ and 3′ ends, respectively. In order to optimize protein expression, Gn and Gc open reading frames (ORFs) (Genbank access No.: MT236316) were subjected to codon optimization based on dogs, and then independently subcloned into the Not I/Pme I sites of another CDV cDNA clone that was constructed previously in our laboratory. Two recombinant plasmids of cDNA clones were separately purified using the PureLinkTM HiPure Plasmid Maxiprep Kit (Thermo Fischer, Carlsbad, USA) according to the manufacturer’s instruction.
Rescue and passaging of rCDV-Gn and -Gc
BSR-T7/5 cells were seeded into a 12-well plate, and cultured at 37 °C with 5% CO2. To rescue recombinant viruses, a cell monolayer at 70% confluency was co-transfected with either of the cDNA clones (2.0 µg/well), pCAGGS-N (1.0 µg/well), pCAGGS-P (0.5 µg/well) and pCAGGS-L (0.5 µg/well) using Lipofectamine 2000 (Thermo Fisher, Carlsbad, USA) according to the manufacturer’s instruction. Two co-transfected cell monolayers were digested with trypsin at 72 h post transfection (hpt), and then separately co-cultivated with VDS cells in two T25 flasks. The rescued viruses were subjected to serial blind passages in VDS cells.
RT-PCR analysis
The rCDV-Gn and-Gc were harvested at passage-10 (P10) for extraction of viral RNAs, which were separately used as templates for RT-PCR analysis using the PrimeScript™ High Fidelity One Step RT-PCR Kit (Takara, Dalian, China). The forward primer (5′-TCAAGAGTATTACTCATGCTTAA-3′) targeted the downstream region of P ORF, and the reverse primer (5′-TCGAAGTCGTACACCTCAGTCAT-3′) targeted the upstream region of M ORF. The RT-PCR reaction underwent 45 °C for 10 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (15 s) and 68 °C (20 s). The extracted RNAs were simultaneously subjected to PCR analysis using the same primers. The PCR reaction contained 2 × PrimeSTAR Max Premix (Takara, Dalian, China) and underwent 30 cycles at 98 °C (10 s), 55 °C (10 s) and 72 °C (10 s). RT-PCR and PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing for analyzing two RT-PCR products.
Indirect immunofluorescence assay (IFA)
Recoveries of rCDV-Gn and-Gc were confirmed by IFA. Briefly, two VDS cell monolayers were independently infected with the P15 rCDV-Gn and -Gc for 24 h, and then fixed in 4% paraformaldehyde at room temperature for 30 min. After fixation, cells were washed four times with PBS, and then permeated with 0.4% Triton X-100 at room temperature for 30 min. After permeation, cells were washed three times with PBS and blocked in blocking solution at 37 °C for 1 h. Subsequently, cells were incubated with the anti-CDV monoclonal antibody (MAb, 1: 400 in blocking solution) at 37 °C for 2 h. After incubation with the primary antibody, cells were washed three times with PBS and incubated with the Alexa Fluor® 555 conjugate (Thermo Fisher, Waltham, MA, USA) (1: 250 in blocking solution) at 37 °C for 1 h. Cells were washed three times with PBS, coated with 90% glycerin, and visualized under the fluorescence microscope. As a control, one non-infected cell monolayer was subjected to the same treatments.
Mass spectrometry
The expressions of Gn and Gc were separately analyzed by mass spectrometry (MS) at the Shanghai Bioprofile Biotechnology Co., Ltd (Shanghai, China), as described previously (Liu et al. 2021). In brief, culture supernatants of P10 progenies were inactivated by 0.1% formalin at 4 °C for 48 h. Protein digestion was performed with a method of filter-aided sample preparation (Wiśniewski et al. 2009). Liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) was performed on a Q Exactive Plus mass spectrometer that was coupled to Easy nLC (Thermo Fisher, Waltham, MA, USA). The MS data were analyzed using MaxQuant software v1.6.0.16. The database search results were filtered and exported with <1% false discovery rate at peptide-spectrum-matched level, and protein level, respectively.
Growth kinetics
The rCDV-Gn and -Gc were compared with each other on their growth kinetics in vitro. Briefly, VDS cells were seeded into five 12-well plates (106 cells/well, and 6 wells/plate) for incubation at 37 °C for 2 h. The P15 rCDV-Gn and -Gc were separately inoculated (MOI = 0.0002) into all plates (3 wells/progeny in each plate) for incubation at 37 °C for 3 h, and then supernatants were replaced with DMEM for further incubation at 37 °C. At 0, 24, 48, 72 and 96 h post inoculation (hpi), any of plates was randomly removed from the incubator, and subjected to two freeze-and-thaw cycles to collect supernatant for viral titration using the Spearman-Kärber equation (Finney 1952). The wt-CDV as a control was subjected to the same treatments. Kinetic curve of virus growth was drawn using the GraphPad Prism software (Version 7.0). Data at each time point were representative of three independent experiments.
Genetic stabilities of two foreign sequences
Two recombinant viruses were subjected to twenty serial passages (3 d/passage) in VDS cells. Their culture supernatants at P15 and P20 were harvested for RT-PCR analysis, as described in Subheading RT-PCR analysis. Two RT-PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing.