Comparison of gE/gI- and TK/gE/gI-Gene-Deleted Pseudorabies Virus Vaccines Mediated by CRISPR/Cas9 and Cre/Lox Systems

Figure 1. The gE/gI-gene-deleted recombinant PRV (rGXΔgE/gI) was developed using CRISPR/Cas9 and Cre/Lox systems. (A,B) Amino acid sequence alignments of PRV gB and gD (PRV GX strain, PRV Bartha strain, and the previous pandemic strain PRV Ea). (C) The vaccine development strategy was performed. PRV virulent gene gE and gI were replaced with mCherry via CRISPR/Cas9 system-assisted homologous recombination. Recombinant virus purification was accelerated using single-cell FACS. The mCherry gene was deleted through the Cre/Lox system. (D) Fluorescence detection was performed when HEK293T cells were co-transfected with pX335-Cas9-sgRNA-gE, pX335-Cas9-sgRNA-gI, gIhm-loxN-CMV-mCherry-SV40polyA-loxN-gEhm donor template and PRV genome. (E) Single cells with fluorescent gene recombinant PRV were selected using FACS. (F) Plaque purification of gE/gI-gene-deleted recombinant PRV in agarose-DMEM plates. (G) Plaque purification for fluorescent gene excision PRV following Cre- recombinase treatment. Arrow indicated cell plaques. (H) PCR verification of gE/gI gene deletion.

Figure 2. The gE/gI/TK-gene-deleted recombinant PRV (rGXΔTK/gE/gI) was developed using CRISPR/Cas9 and Cre/Lox systems. (A) Overview of the strategy for vaccine development. PRV virulent gene TK and gE/gI were replaced with GFP and mCherry respectively via CRISPR/Cas9 system-assisted homologous recombination. Recombinant virus purification was accelerated using single-cell FACS. The GFP and mCherry gene were deleted through the Cre/Lox system. (B) Fluorescence detection was conducted while HEK293T cells were co-transfected with pX335-Cas9-sgRNA-TK, pX335-Cas9-sgRNA-gE, pX335-Cas9-sgRNA-gI, TKhm1-loxP-CMV-GFP-SV40polyA-loxP-TKhm2, and gIhm-loxN-CMV-mCherry-SV40polyA-loxN-gEhm donor templates, PRV genome. Scale bar, 100 μm. (C) Single cells with fluorescent gene recombinant PRV were sorted through FACS. Scale bar, 100 μm. (D) Plaque purification of gE/gI/TK-gene-deleted recombinant PRV in agarose-DMEM plates. Scale bar, 100 μm. (E) Plaque purification for fluorescent genes excision PRV following Cre–recombinase treatment. Arrows indicate cell plaques. Scale bar, 100 μm. (F) PCR verification of TK and gE/gI gene deletion. (G) One-step growth curves of the recombinant viruses in BHK-21 cells.

Figure 3. The effect of the gE/gI- and gE/gI/TK-gene-deleted PRVs on histopathological lesions in mice. (A,B) Pathological lesions of brain and lung tissues were detected using HE staining. Scale bar, 50 or 100 μm. Arrows indicate the cell infiltrates. HE, hematoxylin and eosin staining.

Figure 4. The rGXΔgE/gI or rGXΔTK/gE/gI facilitated production of IFN-γ-producing CD4+ and CD8+ T-cells in the blood. (A) Flow cytometry analysis was used to measure the IFN-γ-producing CD4+ and CD8+ T-cells. (B) The calculation of IFN-γ-producing CD4+ and CD8+ T-cells. n = 4. *** p < 0.001.

Figure 5. PRV-gD-specific IgG titre and neutralizing antibody titre were evaluated. (A) The indirect ELISA was used to detect gD-specific IgG titer from serum samples at 0, 14, 28, 42, 56, and 63 dpi. (B) Neutralizing antibody titer was examined in the serum of the immunized pigs. The neutralizing ability of antisera generated against PRV-GX strain was calculated and presented as the log2 of the reciprocal of the highest serum dilution when PK-15 cell infection was inhibited. n = 4. ns represents not significant.

Figure 6. The effect of rGXΔgE/gI and rGXΔTK/gE/gI on rectal temperature and survival rates in pigs after challenge. Pigs were challenged with virulent PRV GX strain (107 TCID50) at 49 dpi, then (A) rectal temperature was recorded and (B) survival rates were calculated. Rectal temperature ≥40.0 °C was fever. n = 4.

Figure 7. The effect of the gE/gI- and gE/gI/TK-gene-deleted PRV on histopathology in the brain and lung tissues of pigs. (A,B) HE staining was carried out to measure pathological lesions of brain and lung tissues in pigs. Scale bar, 50 or 100 μm. Arrows indicate the cell infiltrates. HE, hematoxylin and eosin staining.