Evaluation of the Deletion of African Swine Fever Virus E111R Gene from the Georgia Isolate in Virus Replication and Virulence in Domestic Pigs

Abstract

African swine fever virus (ASFV) is the causative agent of an often lethal disease in domestic pigs, African swine fever (ASF). ASF is currently a pandemic disease challenging pig production in Eurasia. While the ASFV genome encodes for over 160 proteins, the function of most of them are still not characterized. Among those ASF genes with unknown functions is the E111R gene. It has been recently reported that the deletion of the E111R gene from the genome of the virulent Chinese field isolate SY18 strain produced a reduction of virus virulence when pigs were inoculated at relatively low doses. Conversely, we report here that deletion of the ASFV gene E111R in the Georgia 2010 isolate does not alter the virulence of the parental virus in experimentally inoculated pigs. A recombinant virus lacking the E111R gene, ASFV-G-∆E111R was intramuscularly (IM) inoculated in domestic pigs at a dose of 10² HAD₅₀ of ASFV-G-∆E111R and compared with animals that received a similar dose of virulent ASFV-G. Both, animals inoculated with either the recombinant ASFV-G-∆E111R or the parental virus developed a fatal form of the disease and were euthanized around the 6th-7th day post-inoculation (dpi).

Keywords: ASF; ASFV; ASFV E111R gene; ASFV virulence; African swine fever virus; recombinant virus.

Figure 1

Phylogenetic dynamics of E111R gene in nature. (A) Phylogenetic analysis conducted by the neighbor-joining method using the full-length sequence of the E111R gene indicates the existence of four potential phylogenetic groups. Numbers in parentheses indicate the genotype of different strains based on p72 classification. Percentages of nucleotide (nt) and amino acid (AA) identities within groups are displayed. Pairwise distance analysis showing differences at the nucleotide (B) and amino acid levels (C) between phylogenetic groups are exhibited. (D) Comparison between synonymous (dS) and nonsynonymous (dN) substitution rates during the evolution of the E111R gene. Significant differences between dS and dN were determined by an unpaired t-test. The effect of dS and dN in shaping the phylogenetic relationship among ASFV isolates was assessed by the neighbor-joining method. Trees were reconstructed specifically by either synonymous (E) or nonsynonymous (F) mutations.

Figure 2

Grouping of ASFV isolates based on amino acid similarities in the E111R protein. Amino acid alignment showing similarities in the E111R protein among a group of 17 representative ASFV isolates. ASFV isolates within each box reflect isolates sharing 100% similarity. Five groups were identified. Red numbers in parenthesis indicate the year of collection of specific isolates (in some cases, no information was found). Conservation plot scores reflect the nature of the change at specific sites. Increased scores reflect substitutions between residues with similar biological properties. Analysis was conducted in the software Jalview version 2.11.1.7.

Figure 3

Evolutionary dynamics of the E111R gene in nature. (A) Graphic representation obtained by SLAC analysis, showing the ratio of dN-dS at specific codon sites in the E111R gene of ASFV. Identification of specific codon sites under positive selection (green asterisks) and negative selection (red asterisks) were obtained by FUBAR (posterior probability cutoff value = 0.9) and FEL (cutoff value of p = 0.1). Numbers close to the asterisk indicate the specific codon position. (B) Ancestral reconstruction analysis, showing the evolutionary dynamics of codon 40 of E111R gene. Predicted sequences at internal nodes (most probable common ancestor sequence associated with the divergence between and within genetic groups) and leaf nodes (represented by different isolates). Sequences in blue, green, and orange represent phenotypes (codon/amino acid) AGC/S, ACC/T, and AAC/N, respectively. Analysis was conducted using the mixed effects model of evolution algorithm (MEME) [18]. Results were saved in json format and visualized with the MEME analysis result visualization tool (https://observablehq.com/@spond/meme, accessed 25 April 2024).

Figure 4

Expression profile of the E111R gene of ASFV during in vitro infection of porcine macrophages. Reverse transcription followed by qPCR was used to evaluate the expression profile of the E111R gene during in vitro infection at different time points, up to 24 h. As a reference for this analysis, we used qPCRs to specifically detect the expression of genes encoding ASFV proteins p30 (early expression) and p72 (late expression). Additionally, the β-Actin gene was used as a control to evaluate the quality and levels of RNA during the infection at different time points.

Figure 5

Schematic for the development of ASFV-G-∆E111R. The recombinant vector containing the mCherry reporter gene under the ASFV p72 promoter activity and the gene positions are shown. The nucleotide positions of the area that was deleted in the ASFV-G genome are indicated by the dashed lines. The resulting ASFV-G-∆E111R virus with the cassette inserted is shown on the bottom.

Figure 6

In vitro growth kinetics in primary swine macrophage cell cultures for ASFV-G-∆E111R and parental ASFV-G (MOI = 0.01). Samples were taken from two independent experiments at the indicated time points and titrated. Data represents means and standard deviations of two replicas. Sensitivity using this methodology for detecting virus is ≥log10 1.8 HAD₅₀/mL.

Figure 7

Evolution of body temperature in animals (5 animals/group) IM infected with 10² HAD₅₀ of either ASFV-G-∆E111R or parental ASFV-G.

Figure 8

Evolution of mortality in animals IM infected with 10² HAD₅₀ of either ASFV-G-∆E111R or parental ASFV-G.

Figure 9

Viremia titers detected in pigs IM inoculated with 10² HAD₅₀ of either ASFV-G-∆E111R or ASFV-G. Each symbol represents individual viremia titers in each animal in the groups. Sensitivity of virus detection: ≥log10^1.8 TCID₅₀/mL.