Deletion of African Swine Fever Virus Histone-like Protein, A104R from the Georgia Isolate Drastically Reduces Virus Virulence in Domestic Pigs

Figure 1. Evaluation of the A104R protein across ASFV isolates. (A) Amino acid alignment representing the diversity of A104R protein of ASFV in the field. Residues in white spots represent changes between amino acids with different charge. Conservation plot scores reflect the nature of the change in specific sites, with high scores associated with changes with similar biological properties. Alignment was produced using the software Jalview version 2.11.1.7. (B) Phylogenetic analysis conducted by maximum likelihood method and the Tamura-3 parameter model showing the diversity of A104R gene of ASFV in the field. Based on the cluster distribution, isolates were categorized in four main groups. Numbers above internal branches represent bootstrap values (1000 repetitions). (C) Comparison between the evolutionary rate of synonymous (dS) and nonsynonymous (dN) mutations during the evolution of A104R gene of ASFV. Significant differences between dS and dN rates were calculated by the unpaired T test. (D) The graphic represents the ratio dN-dS at specific codon sites in the A104R gene of ASFV. Red asterisks represent codon sites evolving under purifying selection. Analysis was conducted using the…

Figure 2. Schematic for the development of ASFV-G-∆A104R. The transfer vector contains the p72 promoter and an mCherry cassette; the gene positions are indicated. The homologous arms were designed to have flanking ends to both sides of the deletion/insertion cassette. The nucleotide positions of the area that was deleted in the ASFV-G genome are indicated by the dashed lines. The resulting ASFV-G-∆A104RL virus with the cassette inserted is shown on the bottom.

Figure 3. In vitro growth kinetics in primary swine macrophage cell cultures for ASFV-G-∆A104R and parental ASFV-G (MOI= 0.01). Samples were taken from three independent experiments at the indicated time points and titrated. Data represent means and standard deviations of three replicas. Sensitivity using this methodology for detecting virus is ≥log10 1.8 HAD50/mL. (*) Indicates significant differences.

Figure 4. Evolution of body temperature (A) and mortality (B) in animals (5 animals/group) IM infected with 102 HAD50 of either ASFV-G-∆A104R or parental ASFV-G.

Figure 5. Viremia titers detected in pigs IM inoculated with 102 HAD50 of either ASFV-G-∆A104R (filled symbols), or ASFV-G (empty symbols). Each symbol represents the average of animal titers in each of the groups. Sensitivity of virus detection: >log10 1.8 TCID50/mL.

Figure 6. Anti-ASFV antibody titers detected by ELISA in pigs IM inoculated with 102 HAD50 of ASFV-G-∆A104R. Each point represents values from individual animals.

Figure 7. Evolution of body temperature (A) and mortality (B) in animals (4 animals/group) IM infected with 102 HAD50 of ASFV-G-∆A104R and challenge 28 days later with 102 HAD50 of parental ASFV-G.