Sequential Deletions of Interferon Inhibitors MGF110-9L and MGF505-7R Result in Sterile Immunity against the Eurasia Strain of Africa Swine Fever

FIG 1. Construction and characterization of the two-gene-deleted ASFV-Δ110-9L/505-7R mutant. (A) Schematic demonstration of constructing the ASFV-Δ110-9L/505-7R mutant. Two transfer vectors, namely, pASFV-Δ9L and pASFV-Δ7R, were constructed in the pUC57 backbone by replacing MGF110-9L and MGF505-7R with reporter gene cassettes p72mCherry and p72eGFP, respectively. Nucleotide positions indicative of the boundaries of deletion and replacement relative to parental ASFV CN/GS/2018 genome were labeled. (B) Fluorescent microscopy of BMDM after 48 h infection with the intermittent ASFV-Δ505-7R mutant (upper) and the ASFV-Δ110-9L/505-7R mutant at 0.1 MOI. (C) Schematic of designing 2 pairs of primers located within (centering primer) and outside (flanking primer) of the deleted genes. The expected sizes of PCR products were indicated. (D) Assessment of genetic deletions and replacements by PCR analysis. Viral DNA obtained from parental ASFV or ASFV-Δ110-9L/505-7R mutant were subjected to PCR amplification. In parallel, a pair of primers targeting viral p72 gene was included as an indicator of genome input. (E) Development of triplex rPCR assay for detection and differentiation of…

FIG 2. The ASFV-Δ110-9L/505-7R mutant is highly tolerant to genetic modifications following 30 successive passages. (A) Schematic representation of passage schemes designed for ASFV-Δ110-9L/505-7R mutant. A total of 30 successive passages in vitro were performed. (B) Fluorescent microscopy of cells infected with the initial ASFV-Δ110-9L/505-7R mutant and subsequent viruses at passage 30. (C) Assessment of the stability of simultaneous deletions in the genome of the mutant at passages 5 (P5), 10 (P10), 20 (P20) and 30 (P30) by using triplex rPCR.

FIG 3. Descriptive survival analyses and rectal temperatures of experimental pigs. The animals were primarily inoculated IM with the ASFV-Δ110-9L/505-7R mutant at each indicated doses. At 35 days postinoculation, animals were subsequently exposed to a virulent challenge with an extension of the monitoring period to 21 days. Meanwhile, group of three pigs receiving a lethal dose of parental ASFV was included. (A) Rectal temperatures of three pigs from control group following infection with virulent ASFV. (B) Survival rate of pigs at the period of inoculation. (C) Survival rate of pigs in the virulent challenge. (D) Rectal temperatures of pigs in the virulent challenge.

FIG 4. Viremia of experimental pigs. Groups of five pigs inoculated with increasing titers of ASFV-Δ110-9L/505-7R were challenged i.m. with lethal wild type ASFV. Virus DNA was monitored in the blood collected from pigs during the inoculation period and the challenge period.

FIG 5. High viral loads correlated significantly with tissue abnormalities. (A) The DNA was extracted from tissue samples and subjected to quantification of ASFV genome copy number. Viral loads of tissue samples obtained from unprotected pigs resembled the results from control pigs. The dashed lines indicate the lower limit of detection. (B) Viral loads of tissue samples from totally-protected pigs were significantly diminished. One surviving pig from each of the groups was examined and representatively displayed. SLN, submandibular lymph node; HLN, gastrohepatic lymph node; MLN, mesenteric lymph node. (C and D) Representative figures for tissue pathology in one unprotected and one totally-protected pigs collectively from 103 HAD50-inoculated group as follows: 1, SLN; 2, HLN; 3, MLN; 4, kidney; 5, spleen; 6, lung; 7, heart; and 8, liver.

FIG 6. Comparison of representative microscopic lesions from healthy, unprotected and totally-protected pigs. The latter collectively came from 103 HAD50-inoculated group. Arrow indicates severe acute and diffuse hemorrhages, and large numbers of karyorrhectic cells. Triangles indicate lymphoid depletion and loss of lymphocytes. Stars indicate infiltration of inflammatory cells.

FIG 7. Antibody profile of experimental pigs in the period of inoculation and virulent challenge. Groups of 5 pigs inoculated with increasing titers of ASFV-Δ110-9L/505-7R (103, 104, 105 and 106 HAD50) were challenged i.m. with lethal wild type ASFV (102 HAD50). Anti-ASFV P30-sepcific antibody titers was detected by ELISA. Each curve represents values from individual animals in each group.

FIG 8. Two doses are better than one. (A) Design of animal experiments. In the one-dose inoculation procedure, after a 3-day period of acclimatization, pigs were inoculated with different doses of the ASFV-Δ110-9L/505-7R mutant at day 0. At 35 days postinoculation (dpi), pigs were challenged with 102 HAD50 of highly pathogenic parental ASFV. After extra 21 days postchallenge (dpc), pigs had died or had been euthanized due to ethical reasons. Unlike the one-dose inoculation procedure, pigs received two doses of the mutant at 0 and 14 dpi, respectively. (B) Survival rate of pigs receiving one dose vs two doses at the period of inoculation (left panel) and challenge (right panel). (C) Antibody profile of pigs receiving one dose vs two doses in the period of inoculation and challenge.