Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge

Figures

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  Figure 1. Construction of ASFV-ΔMGF110/360-9L.A, schematic representation of the gene(s) and region(s) deleted in each gene-deleted ASFV. The deleted gene segments were replaced with the p72 eGFP or p72 mCherry reporter gene cassette as indicated. Nucleotide positions indicating the boundaries of the deletion relative to the ASFV CN/GS/2018 genome are indicated. B, fluorescence of virus-infected primary porcine alveolar macrophages. Scale bar represents 300 μm. C, PCR analysis of ASFV-ΔMGF110/360-9L using specific primers targeting MGF110-9L, MGF360-9L, or p72 (B646L) genes. ∗, nonspecific band. D, multistep virus growth curves of ASFV-ΔMGF110-9L, ASFV-ΔMGF360-9L, ASFV-ΔMGF110/360-9L, and parental ASFV CN/GS/2018. Primary swine macrophage cell cultures were infected (MOI = 0.1) with each of the viruses and virus titers at the indicated times postinfection. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. MOI, multiplicity of infection; ASFV, African swine fever virus.

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  Figure 2. Virulence evaluation of ASFV-ΔMGF110/360-9L in pigs.A, body temperature of pigs inoculated with either 104 median hemadsorption units (HAD50) of ASFV CN/GS/2018 (solid lines) or ASFV-ΔMGF110/360-9L (dashed lines). B, survival rates of pigs inoculated with parental ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L. C–F, viral DNA copies in (C) blood, (D) oral swab, (E) nasal swab, or (F) stool swab of pigs. Each shape represents an individual animal from each group. ASFV, African swine fever virus.

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  Figure 3. Protective efficacy of pigs induced by ASFV-ΔMGF110/360-9L. Groups of pigs inoculated with 104 median hemadsorption units (HAD50) of the indicated gene-deleted ASFV were challenged intramuscularly with parental ASFV CN/GS/2018. Samples were collected from dead pigs or surviving pigs that were euthanized on day 13 postchallenge for virus DNA detection. Days postchallenge refers to untreated pigs inoculated with either 104 HAD50 of ASFV CN/GS/2018 or the time after a secondary challenge with 102 HAD50 ASFV CN/GS/2018 from pigs initially challenged with ASFV-ΔMGF110/360-9L for 17 days (shown in Fig. 2). A, body temperature of pigs. B, survival rates of pigs. C–G, viral DNA copies in (C) blood, (D) oral swab, (E) nasal swab, (F) stool swab, or (G) tissues of the pigs, with each shape representing an individual animal from each group. ASFV, African swine fever virus.

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  Figure 4. Detection of ASFV p30 by blocking ELISA.A, anti-p30 antibody detected by blocking ELISA in pigs intramuscularly inoculated with 104 median hemadsorption units (HAD50) of ASFV-ΔMGF110/360-9L or ASFV CN/GS/2018 for 17 days,\ and (B) after secondary challenge with 102 HAD50 of ASFV CN/GS/2018. Each shape represents an individual animal from each group. C, gross pathological changes of heart, liver, spleen, lung, kidney, and lymph nodes in healthy, ASFV challenged and ASFV-ΔMGF110/360-9L vaccinated pigs. MLN, mesenteric lymph node; GLN, gastrohepatic lymph node; SLN, submandibular lymph node. ASFV, African swine fever virus.

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  Figure 5. Transcriptome profiles of primary porcine alveolar macrophages infected with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L (MOI = 0.1) at 12 or 24 h.A, volcano map of differentially expressed genes (DEGs) in PAMs infected with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L. Red dots indicate significantly upregulated DEGs, green dots indicate significantly downregulated DEGs, and gray dots indicate no significant change. B, heat map of DEGs in PAMs infected with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L. C, GO terms at 12 h (top panel) and 24 h (bottom panel). D, KEGG pathway enrichment at 12 h (top panel) and 24 h (bottom panel). E, Tlr2 gene expression of PAMs infected with ASFV CN/GS/2018 (filled bar) or ASFV-ΔMGF110/360-9L (unfilled bar). The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the ASFV and ASFV-ΔMGF110/360-9L groups. ASFV, African swine fever virus; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PAM, porcine alveolar macrophage; MOI, multiplicity of infection; TLR2, Toll-like receptor 2.

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  Figure 6. ASFV-ΔMGF110/360-9L increased Toll-like receptor 2 mRNA level and Pam3CSK4-triggered TLR2 signaling.A, ASFV-ΔMGF110/360-9L-triggered transcription of Tlr2 in primary porcine alveolar macrophages (PAMs). PAMs were infected with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L (MOI = 0.1) for the indicated times followed by RT-PCR analysis. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the ASFV and ASFV-ΔMGF110/360-9L groups. B and C, PAMs were infected with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L (MOI = 0.1) for 12 h or uninfected (mock), and then the cells were treated or not with Pam3CSK4 (100 ng/ml) for 4 h followed by RT-PCR analysis of (B) Il6 or (C) Tnfa expression. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the ASFV- and ASFV-ΔMGF110/360-9L-Pam3CSK4–treated groups. D, effect of ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L infection on IFN-γ–triggered Irf1 transcription in PAMs. PAMs were infected or not with ASFV CN/GS/2018 or ASFV-ΔMGF110/360-9L for 12 h and then treated or not with IFN-γ (100 ng/ml)…

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  Figure 7. Effect of Toll-like receptor 2 on ASFV titer and ASFV p72 protein.A and B, MA-104 cells were transfected with TLR2 plasmid for 24 h, followed by infection with ASFV for the indicated time. A, ASFV titer and (B) ASFV p72 expression level in TLR2-overexpressed MA-104 cells. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the EV- and TLR2-overexpressed groups. C–E, primary porcine alveolar macrophages (PAMs) were transfected with control-RNAi or TLR2-RNAi by Lipofectamine 2000 for 48 h. PAMs were infected with ASFV (MOI = 0.1) for the indicated times followed by RT-PCR analysis. C, TLR2 levels in TLR2-knockdown PAMs infected with ASFV. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the control-RNAi– and TLR2-RNAi–silenced groups. D, ASFV titer of TLR2-knockdown PAM cells. The graphs show the mean ± SD. In the displayed experiment, three replicates were included per experimental condition. ∗p < 0.05, represent the control-RNAi– and TLR2-RNAi–silenced groups. E, ASFV p72 protein in TLR2-knockdown PAMs.…

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  Supporting Figure S1. Sequenced results of ASFV-ΔMGF110/360-9L genome covering the modified regions. ASFV-ΔMGF110/360-9L genome was isolated from virus stock and the modified region was amplified by PCR utilizing primers MGF-360-9L-F/R and MGF-110-9L-F/R. Afterwards, the PCR results were sequenced to confirm the absence of MGF-360-9L gene (A) and MGF-110-9L gene (B).Note: In Fig. S1B, two gaps and one redundant base were found in ASFV-ΔMGF110/360-9L genome. However, the peak map of sequencing result revealed that these results were caused by the sequencing quality. Therefore, these results were corrected by analyzing the peak map of sequencing. After correction, no gaps or mismatches were observed in ASFV-ΔMGF110/360-9L genome.

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  Supporting Figure S2. A, effect of ASFV-ΔMGF360-9L or ASFV-ΔMGF110-9L on Pam3CSK4-triggered phosphorylation of IκBα or p65 in primary porcine alveolar macrophages (PAMs). PAMs were infected with ASFV-ΔMGF360-9L or on Pam3CSK4-triggered phosphorylation of (MOI = 0.1) for 6, 12, and 18 h or uninfected (mock), and then the cells were treated or not with Pam3CSK4 (100 ng/ml) for another 4 h. The cell lysates were analyzed by immunoblotting utilizing the indicated antibodies. B, effect of TLR2 on TLR2 signaling in vitro. The iPAM cells were transfected with HA-TLR2 plasmid (0, 1, 2 and 4 μg) for 20 h, and then treated with Pam3CSK4 (100 ng/ml) for another 4 h. The cell lysates were analyzed by immunoblotting utilizing the indicated antibodies.

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  Supporting Figure S3. Effect of TLR2 overexpression on the replication of ASFV, ASFVΔMGF360-9L, ASFVΔMGF110-9L and ASFVΔMGF110/360-9L.A, MA-104 cells were transfected with TLR2 plasmid for 24 h, and then infected with ASFV, ASFVΔMGF360-9L, ASFVΔMGF110-9L and ASFVΔMGF110/360-9L at a MOI of 0.1 for the indicated time points. Virus titers were measured by HAD50 assay. B, TLR2 overexpression at the indicated time points was analyzed by immunoblotting assay.

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  Supporting Figure S4. Effect of TLR2 knockdown on the replication of ASFV, ASFVΔMGF360-9L, ASFVΔMGF110-9L and ASFVΔMGF110/360-9L.A, PAM cells were silenced with TLR2-RNAi for 48 h, and then infected with ASFV, ASFVΔMGF360-9L, ASFVΔMGF110-9L and ASFVΔMGF110/360-9L at a MOI of 0.1 for the indicated time points. Virus titers were measured by HAD50 assay. B, TLR2 knockdown at the indicated time points was analyzed by immunoblotting assay.