Genotyping and Molecular Characterization of Classical Swine Fever Virus Isolated in China during 2016–2018

Figure 1. Confirmation of CSFV in PK-15 cells by IFA. The figure represented the reaction of anti-E2 MAb 5B8-2 with Shimen, NM16-323, NM16-333, HL18-494 and HL18-490 strains of CSFV. The scale bar is 200 μm.

Figure 2. Phylogenetic tree based on nucleotide sequences of NS5B and E2. (A) Phylogenetic analysis of the 23 new isolates and 46 reference strains based on partial NS5B gene. (B) Phylogenetic analysis of the full-length E2 genes of the 24 new isolates with comparison of 47 reference strains. The trees were constructed using the neighbor-joining (NJ) method, with 1000 bootstrap replicates. The new CSFV isolates were denoted by red diamonds, in which, subgenotype 2.1b isolates were indicated in blue.

Figure 3. Phylogenetic tree based on the complete genome sequences of CSFV strains using the neighbor-joining (NJ) method, with 1000 bootstrap replicates. All new isolates were indicated by red diamonds, in which the subgenotype 2.1d isolates were indicated in pink while subgenotype 2.1b isolates were indicated in blue.

Figure 4. E2 sequence alignment of 24 new CSFV isolates. E2 amino acid sequence alignment of the 24 new CSFV strains isolated in our study, and 24 reference isolates downloaded from NCBI. The B/C domain is indicated by red line, while A/D domain is indicated by blue line and non-antigenic region is indicated by black line. Important epitope regions are also indicated by black line in antigenic regions.

Figure 5. Sequence alignment of amino acids of Erns from 8 new CSFV strains isolated in our study, and 8 reference isolates downloaded from NCBI. Red, blue and purple lines indicate the antigenic regions AR1, AR2 and AR3, respectively. Erns protein residue changes between vaccine strains and others in antigenic regions are indicated by (▲) symbol, residue changes among newly sequenced strains are indicated by (▼) symbols and those between vaccine strains and others in non-antigenic regions are indicated by (■) symbol.

Figure 6. Schematic diagram showing the potential recombination events among different CSFV isolates. The genomic locations and breakpoints of the potential recombining segments are schematically indicated with colored frames. *, the new isolates in this study; **, C-strain vaccine.

Figure 7. One-step growth curve analysis. Growth kinetics of the CSFV HL18-494, NM16-323 and Shimen strains was depicted by one-step growth curve. An MOI of 5 was used to infect PK-15 cells. The cell cultures were harvested at various time points and TCID50 was calculated. The mean values with standard deviations of three independent experiments were calculated. *, p < 0.05; **, p < 0.01, compared with the Shimen strain.