Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus

Figures

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  Figure 1. SFV expression vector system. We used the plasmid pSFVCs-LacZ, which contains the non-structural genome of SFV. To insert heterologous genes into the pSFVCs-LacZ plasmid, LacZ was removed via BamHI restriction endonuclease, EGFP- and p32-, p54-coding ASFV genes were inserted into the vector plasmid. We obtained pSFVCs-EGFP, pSFV-EGFP, pSFV-p32, and pSFV-p54.

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  Figure 2. Expression of heterologous proteins in BHK-21 cells with SFVCs-RP infected. (A) β-galactosidase expression (×200). (B) EGFP expression (×200). (C) Cells were infected with SFVCs-EGFP RPs (×400) for 24 h. CPE was evaluated.

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  Figure 3. Fluorescence signal intensity was compared after removing the viral capsid protein. BHK-21 cells were infected with SFVCs-EGFP and SFV-EGFP RPs for 24 h, and fluorescence signal intensities were compared (×200).

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  Figure 4. IFA and viral growth of ASFV SFV-RPs. (A) IFA of ASFV SFV-RPs infected BHK-21 cells after 60 h (×400). (B) Viral growth in ASFV SFV-RPs-infected BHK-21 cells.

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  Figure 5. Western blotting of ASFV SFV-RPs. ASFV p32 expression in SFV-p32 RP-infected BHK-21 cells (Lane 1), ASFV p54 expression in SFV-p54 RP-infected BHK-21 cells (Lane 2).

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  Figure 6. Comparative weight changes of immunized and non-immunized (PBS group) mice monitored for 35 days after the first immunization. ns, non-significant (P > 0.05).

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  Figure 7. ELISA was used to detect anti-p32 IgG in each group. Cut off calculation: cut off = average value of negative control + 0.15. When the negative control value was ≤ 0.05, it was calculated as 0.05. Positive judgment: samples with OD value ≥ cut off were judged as positive. Negative determination: samples with OD value < cut off were judged as negative. SFV-p32 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE.

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  Figure 8. ELISA was used to detect anti-p54 IgG and calculate the inhibition percentage (IP) in each group. (A) ELISA results for each group. The mean OD of the negative control was ≥ 0.5. (B) Calculation of the IP of all samples in each group. IP of all samples was calculated as follows: IP (%) = (1–OD450 test sample/Mean OD450 negative control)*100. The IP (%) of the positive control was ≥55%. Serum/plasma samples were assessed as follows: IP ≤ 40% (negative), ASF antibodies were absent in the test samples; IP ≥ 50% ASF antibodies were present in the test samples; 50% > IP ≥ 40% doubtable, rechecked in 1–2 weeks. SFV-p54 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE.

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  Figure 9. ELISPOT was used to detect IFN-γ and IL-4 expression in SFV-p32 and -p54 RP-immunized mice. (A) IFN-γ spots of SFV-p32 RP-immunized mice. (B) IL-4 spots of SFV-p32 RP-immunized mice. (C) IFN-γ spots of SFV-p54 RP-immunized mice. (D) IL-4 spots of SFV-p54 RP-immunized mice. For all four panels, 1–5 are the experimental groups and 6, 7, and 8 are the background control, negative control, and positive control, respectively. The numbers indicate the number of spots.