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).