3.5 Comparison of the FPV HNZZ-2 gene sequence with other FPVs
To analyze the molecular characteristics of the FPV HN-ZZ2 strain, primers were designed based on the full gene sequence of the FPV registered in GenBank. The genome sequence of the strain was amplified by PCR (The primer sequence is shown in Table 1) (Qiao et al., 2001; Liu et al., 2001) and the sequencing results were spliced using DNAstar software to obtain a complete genome. Initially, the BLAST suite was used to identify viral sequences based on their similarity to annotated genomes in GenBank. Based on the best hits of blast search, 39 near full-length genomes were selected (Information of reference strain, Table 2). These genomic sequences and the complete genome sequence of the FPV HN-ZZ2 strain were aligned and analyzed. Sequence analysis showed that the nucleotide sequence was highly related to the reference strain, with nucleotide sequence similarity ranging from 98.5% ~99.6%. The nucleotide sequence of FPV HN-ZZ2 strain showed the highest homology with the FPV HH-1/86 strain, previously isolated from Clouded Leopard (99.6%), and the lowest homology with MEV-L strain (98.5%). The bioinformatic analysis showed that the isolate encoded four proteins, including two nonstructural proteins of NS1 (nt154-2160) and NS2 (nt154-423 and 1886-2123), and two structural proteins of VP1 (nt 2160-2197 and 2270-4422) and VP2 (nt 2668-4422) through alternative splicing. Subsequently, a phylogenetic tree for the full nucleotide sequence of the FPV HN-ZZ2 strain and the 39 reference strains was constructed using the Maximum Comprehensive Likelihood. All phylogenetic analyses and tree editions were conducted using the MEGA 7 software. Phylogenetic analysis revealed that FPV HN-ZZ2 clustered together with FPV but was also closely related to the CPV branch (Figure 6).