A Novel Prototype African Swine Fever Virus DIVA (Differentiation Between Infected and Vaccinated Animals) Serological Assay Based on the Detection of Antibodies Against the pEP153R, eGFP, and p72 Proteins

Figures

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  Figure 1. Analysis of the purified recombinant proteins. (a) pEP153R; (b) eGFP. In each case, the left panel shows the analysis by SDS-PAGE with Coomassie staining, and the right panel shows the analysis by immunoblotting using a mouse monoclonal antibody against the tag of each protein. Arrows indicate the band corresponding to the protein of interest. The numbers on the left side of the gels indicate the molecular weight (MW) of each band in the protein ladder (kDa).

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  Figure 2. Analysis of the antigenicity of the target proteins by indirect ELISA: (a) pEP153R, using negative and positive serum samples from a domestic pig infected with Lv17/WB/Rie1; (b) eGFP, analysing negative and positive serum samples from a domestic pig vaccinated with Lv17/WB/Rie1-ΔEP153R. In each graph, the y-axis shows the optical density (OD) obtained in the assay, and the x-axis indicates the antigen coated on the plate. dpi/dpv: days post-infection/vaccination.

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  Figure 3. Interactive dot diagram (ROC analysis) for the indirect ELISAs: (a) ELISA pEP153R; (b) ELISA eGFP. The horizontal line indicates the cut-off value for each assay (S-NC > 0.50 and S-NC > 0.60, respectively). S-NC: sample OD minus the OD of the negative control of the assay.

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  Figure 4. Analysis of the antibody (Ab) response in domestic pigs against the antigens p72 (INgezim® PPA Compac), pEP153R (pEP153R ELISA), and eGFP (eGFP ELISA). (a) Animals infected with Lv17/WB/Rie1; (b) animals vaccinated with different prototype vaccines; (c) data classified by ELISA, from infected (white boxes) or vaccinated (grey boxes) animals. The cut-off of each assay is indicated by a horizontal line. In (a,b), the y-axis shows the percentage of Ab-positive animals in each assay; and in (c), the ELISA blocking percentage or S-NC parameter. In all cases, the x-axis indicates the days post-infection/vaccination (dpi/dpv). The arrows indicate the day on which the animals were challenged (at 30, 35, or 58 dpi/dpv).

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  Figure 5. Analysis of the antibody (Ab) response in wild boar against the antigens p72 (INgezim® PPA Compac), pEP153R (pEP153R ELISA), and eGFP (eGFP ELISA). (a) Animals infected with Lv17/WB/Rie1; (b) animals vaccinated with Lv17/WB/Rie1-∆CD; (c) data classified by ELISA, from infected (white boxes) or vaccinated (grey boxes) animals. The cut-off of each assay is indicated by a horizontal line. In (a,b), the y-axis shows the percentage of Ab-positive animals in each assay; and in (c), the ELISA blocking percentage or S-NC parameter. In all cases, the x-axis indicates the days post-infection/vaccination (dpi/dpv). The arrows indicate the day on which the animals were challenged (30, 42, or 44 dpi/dpv).

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  Figure 6. Antibody (Ab) response of domestic pigs experimentally infected with other attenuated genotype II ASFV isolates, different from the parental virus Lv17/WB/Rie1. The y-axis shows the percentage of Ab-positive animals against the proteins p72 (INgezim® PPA Compac), pEP153R (pEP153R ELISA), or eGFP (eGFP ELISA), and the x-axis shows the days post-infection (dpi).