Construction of a quadruple gene-deleted vaccine confers complete protective immunity against emerging PRV variant challenge in piglets

Fig. 1. Schematic diagrams of the PRV GDFS-delgI/gE/US9/US2 strain. A Diagram of the PRV gI/gE/US9/US2 gene deletion. The transfer plasmid pSK-gIL-US2R was constructed for homologous recombination with co-transfection of two sgRNAs and PRV GDFS genome. B Identification of the plaque-purified viruses by PCR with specific primers (gIF and US2R). PRV GDFS was used as the positive control (P), and DMEM was used as the negative control (N)

Fig. 2. Identification of PRV GDFS-delgI/gE/US9/US2 by immunofluorescence assay. PK-15 cells were infected with either the PRV GDFS strain or the PRV GDFS-delgI/gE/US9/US2 strain and were analyzed by IFA using anti-gB MAb or anti-gE MAb as the primary antibody and FITC-conjugated goat anti-mouse antibody as the secondary antibody. Scale bar indicates 100 μm

Fig. 3. Detection of PRV-specific antibodies in immunized pigs using ELISA. Mean PRV gE-specific antibody responses (A) and PRV gB-specific antibody responses (B) in different groups. ELISA results are expressed as S/N values. Samples with S/N values less than 0.6 were considered positive. Samples with S/N values greater than 0.7 were considered negative. All data are expressed as mean ± SEM. The asterisks (*) indicate statistically significant differences (P < 0.05)

Fig. 4. Detection of neutralizing antibodies in immunized pigs. The neutralizing antibody titers against the PRV GDFS strain (A) or PRV Ea strain (B) were calculated and are expressed as the highest dilutions that resulted in complete inhibition. All data are expressed as mean ± SEM. The asterisk (*) indicates statistically significant differences between different groups (P < 0.05)

Fig. 5. Rectal temperatures (A) and viral shedding (B) of the piglets after PRV GDFS challenge. After PRV challenge, the rectal temperature was recorded, and fever was considered as rectal temperature > 40.5 °C. Viral shedding was detected by viral isolation and expressed as TCID50. All data are expressed as mean ± SEM

Fig. 6. Weight gain of piglets in all groups was recorded after PRV GDFS challenge. Body weights of all pigs were individually measured at 0 DPC (challenge) and 14 DPC (necropsy). All data are ex-pressed as mean ± SEM. The asterisk (*) indicate statistically significant differences between dif-ferent groups (P < 0.05)

Fig. 7. Pathological changes in piglets after PRV GDFS challenge. Autopsy was performed on all dead and surviving piglets. The brains and tonsils were collected from the pigs and subjected to pathological observation. The red arrow represents cerebral vascular hemorrhage in the brain or ulcer of the tonsil

Fig. 8. Histopathological examination of the brains (a–d) and tonsils (e–h) of piglets after PRV GDFS challenge. Original magnification is × 200. The red arrow represents perivascular lymphocyte infiltration and hemorrhage in the brain