Indirect ELISA Using Multi-Antigenic Dominants of VP1, VP2, and VP3 Recombinant Protein to Detect Antibodies Against Senecavirus A in Pigs

Figures

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  Figure 1. Generation, Purification, and Characterization of SVA Target Proteins. (A–C) Antigenicity index of VP1 (A), VP2 (B), and VP3 (C) proteins using the Jameson–Wolf algorithm. Epitope regions are highlighted: VP1 (aa 5–100, 145–177, and 199–261), VP2 (aa2–103, 141–198, and 211–284), and VP3 (aa 7–32, 57–81, 135–153, and 173–236). (D) Antigenic index of the VP2-VP3-VP1 tandem protein. (E) SDS-PAGE analysis of the expressed proteins, showing their size and purity. (F) Western blot confirming the reactivity of the recombinant proteins with SVA-positive swine serum. Lanes: M, protein molecular weight marker; 1, VP1 (~82 kDa); 2, VP2 (~36 kDa); 3, VP3 (~28 kDa); 4, VP2-VP3-VP1 (~73 kDa). The lane order is consistent between (E) and (F), (the original western blot pictures can be found in Figure S1).

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  Figure 2. Establishment of iELISAs for SVA target proteins and evaluation of immunoreactivity. (A–D) Checkerboard titration for determining the antigen coating concentration and secondary antibody dilution for the recombinant VP1, VP2, VP3, and VP2-VP3-VP1 proteins, respectively. Data are presented as heatmaps of the positive-to-negative (P/N) values (generated with GraphPad Prism). For each panel, the optimal condition is marked with an underline. (E) Comparison of the optimal immunoreactivity of the single structural proteins and the tandem protein. Orange and gray bars represent the mean OD450 values (± SD) of SVA-positive and SVA-negative sera (n = 4), respectively, from three independent assays.

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  Figure 3. Development and Standardization of the SVA iELISAs: Optimization and Cut-off Establishment. (A) Screening of serum incubation time (B) Screening of HRP-IgG reaction time (C−F) Diagnostic performance assessment of the rVP2-VP3-VP1 iELISA and rVP2 iELISA. (C,E) Analysis of the ROC curves for both iELISAs is presented, with the AUC serving as an indicator of diagnostic accuracy. (D,F) Dot plots of the sample-to-positive (S/P) ratios for each individual serum sample. The cut-off was defined by maximizing Youden’s index and is represented by a horizontal dashed line in the figure. Student’s t-test evaluated differences, ** p < 0.01, * p < 0.05; ns, not significant.

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  Figure 4. Comparative analysis of sensitivity and specificity between the VP2 and VP2-VP3-VP1 iELISAs. (A) A range from 1:2 to 1:128 of a positive sera for SVA served to establish the analytical sensitivity. (B) Assessment of reaction with positive serum for FMDV (serotype O/A). The left Y-axis represents the FMDV-specific antibody percentage inhibition (obtained from commercial ELISA kits), and the right Y-axis shows the S/P values for the SVA antigens. (C) Cross-reactivity evaluation of the SVA antigens with sera positive for other prevalent pig pathogens (PCV2, PRV, PCV3, PRRSV, CSFV ASFV,). (D) Virus neutralization test (VNT) results for sera from the three inactivated-vaccine immunized pigs. (E,F) Antibody kinetics detected by the iELISAs based on the (E) VP2-VP3-VP1 and (F) VP2 proteins, respectively. Across figures (A–F), the dashed lines are defined by the established cut-offs specific to each assay.

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  Figure 5. Seroepidemiological of SVA in China (2023−2024). (A) Schematic map illustrating the quantity and geographical distribution of the 3851 pig serum samples across Mainland China. Legend: Dark red, >1000 serum samples; light red, 1–1000 serum samples; white, 0 serum samples. (B) Annual SVA antibody seroprevalence from 2023 to 2024. (C) Provincial SVA antibody seroprevalence across 20 provinces and the overall rate. (D) Seasonal dynamics of SVA seroprevalence. (E) SVA seroprevalence among different swine groups.