Viruses, cell lines, and clinical samples
The vaccine strain CDV-3 (GenBank accession number: EU726268.1), wild-type strain CDV-PS (GenBank accession number: JN896331.1), and the Vero-dslam cell line expressing canine Signaling lymphocyte activation molecule (SLAM) was preserved in our laboratory.
The CDV-positive clinical specimens were collected from the Idog pet hospitals in Changchun, Jilin, China. Diseased dogs show clinical signs of respiratory, nervous and digestive, systems. After a natural death, an autopsy is performed at the hospital. Fresh tissues including lung, spleen, liver, and lymph nodes were collected for laboratory diagnosis and virus isolation.
Virus isolation
The CDV positive lung sample was inoculated into the Vero-dslam cell line for virus isolation. First, CDV positive lung tissue homogenate was prepared in Dulbecco Modified Eagle medium (DMEM) serum-free medium. After ultrasonic treatment and centrifugation of the samples,the viral suspension was inoculated into 90% monolayer Vero-dslam cells in 6-well plates. The Vero-dslam cells inoculated viral suspension were maintained in DMEM containing 5% fetal calf serum (FCS), 100 µg/ml streptomycin and 100 units/ml penicillin. Replacement fresh medium in the culture wells, after 30 min incubation. The Vero-dslam cells were placed in a constant and humidified 35°C incubator with 5% CO2. The cell cultures were observed and recorded daily for the appearance of cytopathic effect (CPE) for 6 days. When obvious CPE apparent in the Vero-dslam cells were observed under an inverted microscope, the viral cultures was collected. The titer of the CDV was determined by 50% tissue culture infective dose (TCID 50) assay and calculated by Reed and Muench method. The remaining viruses were stored at −80°C for use.
Immunofluorescence Assay (IFA)
Monoclonal antibodies (mAbs) C8 against CDV N protein and mAbs 1A5 against CDV H protein were preserved in our laboratory [2, 24]. The Vero-dslam cells were seeded in the 96-well plates, and the cells infected by the CDV isolate were used as experimental group, and the cells without CDV infection were used as the negative control group. At 36 h after infection, all groups of Vero-dslam cells were fixed with 100 µL ice-cold cell fixation solution (4% paraformaldehyde and 0.1% Triton X-100 in PBS) for 60 min; then the fixation solution was removed, the fixed cells were washed twice by using PBS. The fixed Vero-dslam cells were blocked with 5% skim milk in TBST at 37°C for 60-120 min. After blocking, all groups of cells were incubated with the primary antibodies mAbs C8 and 1A5 at 37°C for 120 min, respectively. Then, all cells were washed by using PBST three times and incubated with secondary antibodise Dylight594–conjugated goat antimouse IgG (Abbkine, China) at 37°C for 60-90 min. After the incubation, all groups of cells were washed three times by using PBST. Finally, all groups of Vero-dslam cells were observed by fluorescence microscope (Nikon TS100, Japan). Since the Vero-dslam cell line used in this research expressed the Enhanced Green Fluorescent Protein (EGFP) and could emit green fluorescence, we selected Dylight594-conjugated goat anti mouse IgG which could emit orange-red fluorescence for the IFA.
RNA extraction and reverse-transcription (RT)
The viral genome RNA was extracted by using Easypure® Viral RNA Kit (TransGen, Beijing, China). RT of viral genomic RNA was performed by using PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Reverse-transcribed reaction system (20-μL volumes) containing Template RNA <1 μg, PrimeScript RTase (200 U/μL) 1.0 μL, RNase Inhibitor (40 U/μL) 0.5 μL, Random Primers (50 μM) 1.0 μL, dNTP Mixture (10 mM each) 1.0 μL, 5× PrimeScript Buffer 4.0 μL, RNase free dH2O up to 20 μL. The reaction system was placed at 42°C for 30-60 min, and then at 95°C for 5 min.
Primers, amplification, cloning, and sequencing of the genome
Fourteen pairs of primers covering the whole CDV genome were designed by Primer 5.0 software based on the conserved region of CDV genome (Table 1). The amplification conditions for the PCR reactions were performed by three steps, First, initial denaturation at 95°C for 5 min. Second, the target fragment was amplified by 35 cycles of denaturation at 95°C for 30 s, annealing at 50–58°C for 60–120 s and extension at 72°C for 30 s. Finally, the final extension is performed at 72°C for 8 min. The PCR products with the expected size were purified and ligated into the pMD 18-T vector (TaKaRa, Dalian, China). For each fragment, 3 positive recombinant plasmids were sequenced using M13 universal primers by Comate Bioscience Company (Changchun, China). Sequences are processed and assembled by using the Lasergene program package (DNAStar, Madison, WI, USA).
Phylogenetic assay
The entire H protein Coding sequence (CDS) and full genome sequence were aligned with the sequences of other CDV strains in the GenBank collected from different places and years worldwide respectively. Subsequently, the Maximum-likelihood analyses were performed by general time reversible (GTR+G+I) model with 1000 bootstraps in MEGA 6.0 [25].
Western Blotting (WB)
The antigenic differences between the CDV isolate, CDV-PS and CDV3 were analyzed by WB assay using two mAbs C8 and 1A5. Virus samples was separated by 12% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). And then the virus proteins were transferred to 0.45 μm PVDF membranes (Merck Millipore, Germany) by wet-blotting.
The PVDF membranes were blocked at 37°C for 120 min in 5% skim milk in TBST, and then incubated with the mAbs C8 and 1A5 (diluted 1:2,000 in 5% skim milk in TBST) at 37°C for 90-120 min, respectively. Subsequently,all PVDF membranes were washed three times by using TBST and then incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG (CWBIO, China) at 37°C for 60 min. After incubating secondary antibodies, the membranes were washed there times once again using TBST. Finally, the color rendering was performed by using BeyoECL Star kit (Beyotime, China).
Analysis of epitope information
To determine the homology of the epitopes between different CDV strains, the amino acid sequences of the region encompassing the mAbs C8 and 1A5 truncated B-cell epitopes were aligned with other geographic lineages CDV strains which were available in the NCBI database (https://www.ncbi.nlm.nih.gov/protein/). The sequences of CDV genome containing the coding region information of all structural proteins of CDV were selected. Sequences are processed and edited by using the Lasergene program package (DNAStar, Madison, WI, USA).