First Isolation and Characterization of Three Strains of Porcine Sapelovirus in Yunnan Province, China

Figures

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  Figure 1. The CPEs, TEM and plaque formation of the PSV-ML-15 strain. (A) The CPEs of PSV-ML-15 in IPEC-J2 (scale bar = 100 µm). (B) The CPEs of PSV-ML-15 in PK-15 (scale bar = 100 µm). (C) The CPEs of PSV-ML-15 in ST (scale bar = 100 µm). (D) The cell control of IPEC-J2 (scale bar = 100 µm). (E) The cell control of PK-15 (scale bar = 100 µm). (F) The cell control of ST (scale bar = 100 µm). (G) Transmission electron microscopic image of purified PSV particles. (H,I) Plaque formation of PSV-ML-15 in PK-15 cells (stained with crystal violet at 72 hpi).

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  Figure 2. Growth kinetics of PSV-ML-15 in different cell lines with an MOI of 0.01.

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  Figure 3. Heatmap of nucleotide identity between PSVs isolated in this study and reference PSV strains. The genomes of 136 PSV strains were divided into 12 sequential genes (L, VP4, VP2, VP3, VP1, 2A to 2C, 3A to 3D), and each gene of reference PSVs was compared with the synonymous gene of the three isolated PSVs in this study; the sequence identity between every pair was measured and represented as a heatmap, scaled such that the minimum inter-sequence identity (62.1%) is displayed as red and the maximum inter-sequence identity (100%) as blue.

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  Figure 4. Heatmap of amino acid identity between PSVs isolated in this study and reference PSV strains. The polyproteins of 136 PSV strains were divided into 12 sequential protein (L, VP4, VP2, VP3, VP1, 2A to 2C, 3A to 3D), and each protein of reference PSVs was compared with the synonymous protein of the three isolated PSVs in this study; the sequence identity between every pair was measured and represented as a heatmap, scaled such that the minimum inter-sequence identity (62.5%) is displayed as red and the maximum inter-sequence identity (100%) as blue.

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  Figure 5. Phylogenetic analysis based on the genomes of PSV strains. The tree was constructed by the neighbor-joining method with MEGA v.6.0. The numbers on the branches are bootstrap values (percent) from 1000 replicates. The three PSV strains isolated in this study are marked with a red dot, and the sequences of other representative PSVs are indicated as ‘GenBank accession number+PSV-1+Strain number+Country+Year’.

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  Figure 6. Phylogenetic analysis based on the nucleotide sequence of 3D gene. The tree was constructed by the neighbor-joining method with MEGA v.6.0. The numbers on the branches are bootstrap values (percent) from 1000 replicates. The three PSV strains isolated in this study are marked with a red dot, and the sequences of other representative PSVs are indicated as ‘GenBank accession number+PSV-1+Strain number+Country+Year’.

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  Figure 7. Phylogenetic analysis based on the nucleotide sequence of VP1 gene. The tree was constructed by the neighbor-joining method with MEGA v.6.0. The numbers on the branches are bootstrap values (percent) from 1000 replicates. The three PSV strains isolated in this study are marked with a red dot, and the sequences of other representative PSVs are indicated as ‘GenBank accession number+PSV-1+Strain number+Country+Year’.

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  Figure 8. Genome recombination analyses of PSV-ML-19. (A) The genome schematic diagram of PSV: the red area indicates the region of origin from PSV-21-V, and the blue area indicates the region of origin from PSV GER L00798-K11 14-02. (B) Similarity plot analysis of the genome sequences of PSV-21-V (red curve), GER L00798-K11 14-02 (blue curve) and PSV-ML-19 as query sequences; the y-axis presents the percent identity with a window size of 200 bp and a step size of 20 bp. The vertical purple line indicates the potential recombination breakpoint. (C) The recombination breakpoint analysis of PSV-21-V vs. PSV GER L00798-K11 14-02 (yellow curve), PSV-21-V vs. PSV-ML-15 (light green curve), and PSV GER L00798-K11 14-02 vs. PSV-ML-15 (purple curve). (D) The phylogenetic tree constructed by the neighbor-joining method in MEGA 6.06 based on the concatenated sequences of the major parent. (E) The phylogenetic tree constructed by the neighbor-joining method in MEGA 6.06 based on the sequences of the minor parent.