Recombinant pseudorabies virus expressing the consensus VP2 protein of porcine parvovirus 1 (PPV1) protects pigs against pseudorabies virus and PPV1

Figures

• 

  Figure 1. Construction and characterization of PRV-ΔUL39/TK/gE/gI. A Schematic diagram of the PRV-ΔUL39/TK/gE/gI construction strategy. B Polymerase chain reaction (PCR) identification of the deletions of the UL39, TK, gE and gI genes. Lines 1 and 2 represent PRV HLJ8 and PRV-ΔUL39/TK/gE/gI, respectively. C Plaque morphology of PRV HLJ8 and PRV-ΔUL39/TK/gE/gI in Vero cells. D Comparison of plaque sizes between PRV HLJ8 and PRV-ΔUL39/TK/gE/gI. Fifty plaques for each strain were randomly chosen to calculate plaque size. The data are presented as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test (****, p < 0.0001). E One-step growth curves of PRV-ΔUL39/TK/gE/gI and PRV HLJ8 in Vero cells.

• 

  Figure 2. Safety and immunogenicity evaluation of PRV-ΔUL39/TK/gE/gI in pigs. A Schematic of the PRV-ΔUL39/TK/gE/gI immunization procedure in pigs. B Survival rate of pigs immunized with PRV-ΔUL39/TK/gE/gI. C PRV gB-specific Ab levels in pigs immunized with PRV-ΔUL39/TK/gE/gI. D PRV gE-specific Ab levels in pigs immunized with PRV-ΔUL39/TK/gE/gI. E Viral loads in serum at 0, 7, 14, and 21 dpi. F Viral load in tissues detected by qPCR.

• 

  Figure 3. Construction and identification of rPRV-VP2. A Schematic diagram of the rPRV-VP2 construction strategy. B Plaque purification of the recombinant virus. Virus plaques are indicated with red arrows. Scale bars: 200 μm. C PCR identification of the insertion of the VP2 gene and deletion of virulence genes. Lines 1 and 2 represent rPRV-VP2 and PRV HLJ8, respectively. D Western blotting (WB) analysis of the expression of the VP2 protein in Vero cells infected with rPRV-VP2. Lines 1–3 represent rPRV-VP2-, PRV HLJ8-, and mock-infected cells, respectively. E Detection of the expression of the PPV1 VP2 and PRV gB proteins by an immunofluorescence assay (IFA). Scale bars: 200 μ.

• 

  Figure 4. Growth properties, genetic stability and hemagglutination activity of rPRV-VP2. A One-step growth curves of PRV HLJ8 and rPRV-VP2 in Vero cells. B Plaque morphology of PRV HLJ8 and rPRV-VP2 in Vero cells. C Comparison of plaque sizes between PRV HLJ8 and rPRV-VP2. Fifty plaques for each strain were randomly chosen to calculate plaque size. The data are presented as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test (****, p < 0.0001). D PCR identification of the inserted target gene (VP2) in cell cultures from generations 1, 5, 10, 15, and 20 of rPRV-VP2. 1–5 represent the 1st, 5th, 10th, 15th, and 20th passages of rPRV-VP2, respectively, whereas 6 represents the negative control (ddH2O). (E) Haemagglutination test of rPRV-VP2.

• 

  Figure 5. Immunological protective effect of rPRV-VP2 in mice. A Schematic of the rPRV-VP2 immunization procedure in mice. B PRV gB-specific Ab levels in mice immunized with rPRV-VP2. C PPV1 VP2-specific Ab levels in mice immunized with rPRV-VP2. D Body weight changes in mice after immunization with rPRV-VP2. E Survival curves of mice following challenge with PRV HLJ8.

• 

  Figure 6. Protection of pigs vaccinated with rPRV-VP2 against PRV challenge. A Schematic of the rPRV-VP2 immunization procedure in pigs. TP is a commercial live attenuated vaccine, and JS-2012-△gE/gI is a commercial inactivated vaccine. B PRV gB-specific Ab levels in pigs immunized with rPRV-VP2. The TP group was immunized once, coinciding with the booster vaccination administered to the other groups (21 dpi, indicated by an arrow). Statistical significance between the TP group and each of the rPRV-VP2 and inactivated vaccine groups was analysed by two-way ANOVA (**, p < 0.01). C PRV gE-specific Ab levels in pigs immunized with rPRV-VP2. Rectal temperature changes (D), clinical symptoms (E), daily weight gain rate (F), and survival curves (G) of pigs following challenge with PRV HLJ8. (H) Viral load in serum at different time points post-challenge. I Viral load in tissues detected by qPCR.

• 

  Figure 7. Observation of pathological changes in piglet tissues after PRV challenge. Piglets in the DMEM control group exhibited extensive haemorrhage and congestion in the lungs, brain, tonsils, and submandibular lymph nodes. In contrast, those in the immunized group presented intact organ structures with normal colour and texture and no pathological alterations.

• 

  Figure 8. Histopathological changes in piglet tissues after PRV challenge. The bar representing the histological images of the lesions represents 50 μm.

• 

  Figure 9. Protection of pigs vaccinated with rPRV-VP2 against PPV1 challenge. A Schematic of the rPRV-VP2 immunization procedure in pigs. B PPV1 VP2-specific Ab levels in pigs immunized with rPRV-VP2. C HI antibody levels in pig sera after immunization. The daily weight gain rate (D) and rectal temperature (E) were monitored for the indicated times. F Viral load in tissues detected by qPCR.