*2.2. Phylogenetic Analysis of the E2 Gene*

The nucleotide sequences of the three isolated CSFV strains were compared with those of the other CSFV strains in GenBank. Sequence analysis revealed that the E2 gene of the three isolated strains showed 81.1–98.4% nucleotide similarity with other Chinese strains. The E2 AA sequence homologies among three isolated strains ranged from 95.6% to 99.5% and all isolates shared high similarities of 96.4–99.7% with CSFV from subgenotype 2.1 and lower identities with genotype 1 (89.3–90.4%).

The phylogenetic trees of the full-length E2 gene were constructed by using MEGA7.0 (https:// www.megasoftware.net/). Phylogenetic analysis indicated that the three isolated CSFV strains belonged to subgenotype 2.1. CSFV HBXY-2019 and GXGG-2019 isolates were grouped into subgenotype 2.1d and GDGZ-2019 was grouped into subgenotype 2.1c (Figure 2).

**Figure 2.** Phylogenetic analysis based on the full-length E2 gene of the isolated virus. Phylogenetic trees were constructed using MEGA 7.0.18 software with the neighbor-joining method (1000 bootstrap replicates). The isolated virus is marked in red.

#### *2.3. Comparison of the AA Mutations of the E2 Gene in Three CSFV Isolated Strains*

As shown in Figure 3, 44 mutated AAs were observed between the three CSFV isolated strains and subgenotype 1.1, and 15 mutated AAs were detected among GDGZ-2019, HBXY-2019 and GXGG-2019. Two AAs were mutated in HBXY-2019 and GXGG-2019 (K197M and R303K). The E2 proteins of different genotypes contained high levels of mutated AAs, indicating that E2 proteins were mutant viral proteins (Figure 3 and Table 1).
