Detection of CPV-2
A pair of primers were designed to detect CPV-2. The forward primer is CTGTGGGTAATGTTGGTTGTT (5’to 3’), and the reverse primer is TGGTCTTGATGTTGATGGATG (5’ to 3’). The product’s length is 1163bp. We used DNAiso Reagent (Takara, China) to extract the genome and ExTaq DNA Polymerase (Takara, China) to amplify the gene. The following amplification procedure was applied: pre-denaturation at 94 °C for 5 min, then 35 cycles of denaturation at 94 °C for 30 sec, annealing at 48 °C for 30 sec and extension at 72 °C for 1 min. The final extension at 72 °C for 7 min was performed after the cycles.
Amplification and sequencing of VP2 gene
Another pair of primers were designed to amplify the whole VP2 gene. The forward primer is CACCAGATCATCCATCAACATC (5’ to 3’), and the reverse primer is AACCACCCACACCATAAC (5’ to 3’). The product is 2293bp in length encompassing the entire VP2 gene. The super-fidelity DNA Polymerase used was 2 × Phanta Max Master Mix (Vazyme, China). Amplification conditions were performed following the manufacturer’s instructions: pre-denaturation at 95 °C for 30 sec, then 35 cycles of denaturation at 95 °C for 15 sec, annealing at 56 °C for 15 sec and extension at 72 °C for 1.5 min, and a final extension at 72 °C for 5 min.
The PCR product was purified by EasyPure Quick Gel Extraction Kit (TransGen, China) according to the manufacturer’s instructions. Then it was cloned into pEASY-Blunt Cloning Vector (TransGen, China). The recombinant vector was transformed into Trans1-T1 Phage Resistant Chemically Competent Cell (TransGen, China). Positive clones were screened by blue-white selection and further verified by the PCR test, thus were sent to a third-party company (Huada Gene, Beijing) for sequencing.
SequenceAnalysis and Phylogenetic Construction
Sequences were assembled using Seqman (DNASTAR, USA), then the nucleotide sequences and deduced amino acid sequences were aligned with the ClustalW method. To construct a nucleotide phylogenetic tree based on the full VP2 gene, MEGA-X was used to find the best DNA models with maximum likelihood (ML) method. The bootstrap values were 1000 to analyze the confidence level. All strains were analyzed with reference strains obtained from Genbank. Three CPV-2 vaccine strains (Genbank accession number: FJ197847, FJ011098, and FJ011097) and two FPLV vaccine strains (Genbank accession number: EU498681 and EU498680) were included.
Temporal and Geographical Distribution Analysis
The reported Chinese CPV-2 strains between 2014 to 2019 with clear background were collected either from Genbank or related publications, along with CPV-2 strains in the study (the CPV-2 strains collected in 2020 were excluded since no other CPV-2 strains were isolated in 2020 currently). These data were subjected to temporal distribution analysis and geographical distribution analysis.