Deletion of the ASFV dUTPase Gene E165R from the Genome of Highly Virulent African Swine Fever Virus Georgia 2010 Does Not Affect Virus Replication or Virulence in Domestic Pigs

Figure 1. Evaluation of the E165R across ASFV isolates. (A) Amino-acid alignment representing the diversity of E165R protein of ASFV in the field. Residues in white spots represent changes between amino acids with different charges. Conservation plot scores reflect the nature of the change in specific sites: high scores are 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 Kimura-2 parameter model showing the diversity of E165R 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) The graphic represents the ratio dN-dS at specific codon sites in the E165R gene of ASFV. Red asterisks represent codon sites evolving under purifying selection, and green asterisks represent codons evolving under positive selection. Analyses were conducted using the evolutionary algorithms FEL and MEME considering a cutoff value of p = 0.1.

Figure 2. Schematic for the development of ASFV-G-∆E165R. The recombinant 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-∆E165R 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-∆E1654R and parental ASFV-G (MOI = 0.01). Samples were taken from three independent experiments at the indicated time points and titrated in swine macrophages. Data represent means and standard deviations of three replicas. Sensitivity using this methodology for detecting virus is ≥log10 1.8 HAD50/mL.

Figure 4. Evolution of body temperature in animals (5 animals/group) IM infected with 102 HAD50 of either ASFV-G-∆E165R or parental ASFV-G. Unpaired t test with Welch correction using the Two-stage step-up (Benjamini, Krieger, and Yekutieli) method was conducted to assess statistical differences in temperatures between ASFV-G and ASFV-G-ΔE165R at different days post-infection. Significant differences between groups found at days four and five post-infection were evaluated using the false discovery rate method (FDR), being p-values < 0.05 considered significant.

Figure 5. Evolution of mortality in animals (5 animals/group) IM infected with 102 HAD50 of either ASFV-G-∆E165R or parental ASFV-G. Significant statistical differences were predicted between the two groups of pigs when evaluated by both the Log-rank (Mantel-Cox) test (p-value 0.008) and Gehan–Breslow–Wilcoxon test (p-value 0.009).

Figure 6. Viremia titers detected in pigs IM inoculated with 102 HAD50 of either ASFV-G-∆E165R, or ASFV-G. Each curve represents individual animal titers. Sensitivity of virus detection: > log101.8 TCID50/mL. Significant differences in viremia titers between the two groups were detected at day four post infection, calculated using the unpaired t-test considering the two-stage step-up (Benjamini, Krieger, and Yekutieli) method. The significance was evaluated using the false discovery rate method (FDR), being p-values < 0.05 considered significant. All calculations were conducted using the software Graphpad Prism version 9.2.0.