Evaluation of African Swine Fever Virus E111R Gene on Viral Replication and Porcine Virulence

Figure 1. ASFV SY18ΔE111R construction and in vitro growth characteristics. (a) Schematic diagram of SY18ΔE111R recombinant virus construction. (b) The PCR amplification results of the E111R gene of genomic DNA. M, DL2000 DNA Marker; (1) PCR product of the E111R gene of the 1st-generation SY18ΔE111R strain; (2) PCR product of the E111R gene of the 2nd-generation SY18ΔE111R strain; (3) PCR product of the E111R gene of the ASFV SY18 strain; (4) negative control. (c) PAMs were infected by 2nd generation SY18ΔE111R after 96 h (20×, scale bar 50 μm). The 2nd generation SY18ΔE111R infected cells express green fluorescence (left), bright field plot of the 2nd generation SY18ΔE111R strain-infected cells (right). (d) In vitro growth characteristics of the 2nd generation SY18ΔE111R and ASFV SY18. Both viruses infected PAMs at 0.01 MOI and titrated for viral yield at 2, 6, 12, 24, 48, 72, 96, and 120 hpi; data representing the mean of the three independent experiments’ results are expressed as log10 TCID50/mL. Data are not significantly different for all time points (p > 0.05).

Figure 2. Comparison of E111R amino acid sequences in 16 different ASFV isolates. The E111R of ASFV SY18 was used as the reference sequence in the comparison, where single-letter abbreviations indicate amino acid sequences. Different letters indicate other amino acids; the same amino acid is shown as “.”. The amino acid homology of pE111R between isolates is 95.5% to 100%.

Figure 3. E111R gene transcript expression results. The graph shows the results of the expression of the E111R, CP204L, and B646L genes in the ASFV genome relative to the GAPDH gene. The RNA was collected from PAMs at 1, 4, 8, 12, 16, and 24 h after infection with ASFV (MOI = 3). Transcript levels of p30, p72, and E111R genes after ASFV SY18 (MOI = 3) infection of PAMs are indicated by a dash (circle marker), and data from the parallel treatment group treated with cytarabine (AraC, 100 μg/mL) are shown as squares. The Y-axis uses the decimal (log10) logarithm to indicate the relative mRNA expression levels.

Figure 4. Results of survival rates, rectal temperature, and p54 antibody S/P values in pigs. (a) Survival rates of pigs infected with ASFV SY18 and SY18ΔE111R. (b) Rectal temperature data in pigs after infection. Each line represents the body temperature data of an individual animal. (c) The p54 antibody S/P values of animal sera. Values of S(OD450)/P(OD450) greater than 0.25 for each group were considered positive.

Figure 5. Viral load results for tissues. Black dots represent the viral load of individuals in the tissue.