Identification and Isolation of Two Different Subpopulations Within African Swine Fever Virus Arm/07 Stock

Figures

• 

  Figure 1. Allele balance distribution of variants from the Arm/07 stock along the Georgia 2007/1 reference genome.

• 

  Figure 2. Genome assembly of Arm/07/CBM/c2. (A) Circular representation of the genome indicating predicted CDS in “forward” (blue) or “reverse” (red) orientation in the outer circle and the GC/AT content (purple indicates higher AT content, green indicates higher GC content) in the inner circle. (B) Whole genome coverage plot using Illumina sequencing reads mapped to the ASFV Arm/07/CBM/c2 assembly.

• 

  Figure 3. Genome assembly of Arm/07/CBM/c4. (A) Whole genome coverage plot using Illumina and Nanopore (ONT) sequencing reads mapped to the ASFV Arm/07 isolate clone 4 assembly. (B) Allele balance distribution of variants from Illumina reads along the Arm/07/CBM/c4 assembly.

• 

  Figure 4. Phylogenetic tree of different ASFV genotypes built by maximum-likelihood method using sequences corresponding to the central conserved region (CCR, comprising 129 kb between A224L and I196L genes). Arm/07/CBM/c2 (highlighted in red) is grouped within the genotype II strains while Arm/07/CBM/c4 (highlighted in blue) is grouped within the genotype I strains. The tree was built based on MAFFT aligned genomes using IqTree software. Automatic settings were used, resulting in the selection of HKY+FK3PU+F+R2 as the best fit model; statistical support of the shown branches was obtained with an ultrafast bootstrap repetition value of 10,000. Substitution per base rate is indicated in the scale below.

• 

  Figure 5. Recombination events inferred by RDP (Recombination Detection Program) in the ASFV genomes used for the phylogenetic tree construction.

• 

  Figure 6. Multiple nucleotide (A) and amino acid alignment (B) of EP402R sequences corresponding to the genotype I strains numbered 1–9 and Armenia/07/CBM/c4 strain performed using ClustalW. A: Nucleotide alignment of EP402R sequences show that ORF in strains 1–4 and 6 are codified in the third reading frame (ATG in red) whereas ORF for strain 5 and 8–10 is codified in second reading frame (ATG in blue). T deletion identified in Arm/07/CBM/c4 is highlighted by a red square. B: Amino acid alignment of EP402R sequences corresponding to the indicated strains.

• 

  Figure 7. Arm/07/CBM/c2 and Arm/07/CBM/c4 clones express functional CD2v and have a similar capacity for growth in PAMs. PAMs were mock-infected or infected with either Arm/07/CBM/c2 or Arm/07/CBM/c4 (MOI = 0.5) for 16 h. Then, (A) porcine erythrocytes were added and after 24 h, rosettes (blue arrows) were detected on a light microscope. (B) cells were lysed in RIPA buffer and lysates were separated by 7–20% SDS-PAGE, followed by immunoblotting with anti-CD2v, anti-p72, anti-p32 ASFV proteins or anti-actin. PAMs (C) or COS cells (D) were infected with either Arm/07/CBM/c2 or Arm/07/CBM/c4 for 24, 48 or 72 h and the total (TV) and extracellular (EV) virus was then assayed for HAD activity. Scale bar in A represents 200 µm.

• 

  Figure 8. Arm/07/CBM/c2 but not Arm/07/CBM/c4, prevented STING and IRF3 phosphorylation in PAM. PAMs were mock-infected or infected with either Arm/07/CBM/c2, Arm/07/CBM/c4 or NH/P68 strains for 16 h using MOI = 0.5. Cells were lysed in RIPA buffer and lysates were separated by 7–20% SDS-PAGE, followed by immunoblotting with anti-pSTING, anti-p-IRF3, anti-p32 and anti-Actin.