Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013

Fig. 1. Construction strategy of the PRV-GD2013-ΔgI/gE recombinant strain. a, b Position of the left and right homologous recombination arms (L-arm and R-arm). c, d The genome of PRV-GD2013 and the relative site of US6 (gD), US7 (gI), US8 (gE), US9, and US2. e Construction of the transfer plasmids pBLE-gI-gE, including the target deletion region, L-arm, and R-arm. f Construction of the transfer plasmids pBLE-gI-EGFP-gE, including the target deletion region, L-arm, R-arm, and inserted EGFP expression cassette. g The genome of PRV-GD2013-ΔgI/gE-EGFP and the relative site of US6, US7s, the EGFP expression cassette, US8s, US9, and US2. h The genome of PRV-GD2013-ΔgI/gE and the relative site of US6, US7s, US8s, US9, and US2. UL, unique long region; US, unique short region; IR, internal repeat sequences; TR, terminal repeat sequences; US7s, section US7; US8s, section US8. EGFP expression cassette; EGFP (enhanced green fluorescent protein)

Fig. 2. BHK-21 cells were infected with recombinant PRV. CPEs caused by PRV-GD2013-ΔgI/gE (a), PRV-GD2013-ΔgI/gE-EGFP (b), PRV-GD2013 (c), and control (d). CPEs were observed using a fluorescent microscope at two dpi

Fig. 3. a One-step growth curve of PRV-GD2013-ΔgI/gE compared with its parental viruses. Monolayers of BHK-21 cells were inoculated with PRV-GD2013-ΔgI/gE and PRV-GD2013 at 1 MOI. The cell culture supernatants were collected at different time points (0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 hpi), and were used to calculate the TCID50 of each virus. b Plaque morphology and plaque size measurement of PRV-GD2013-ΔgI/gE and PRV-GD2013 on BHK-21 cells at 60 h post-infection. The one-step growth curve and plaque size were measured by one-way repeated measurement analysis variance and least significance (LSD). Differences were considered statistically significant when p < 0.05

Fig. 4. Pathogenic testing in piglets using recombinant PRVs. a Rectal temperature of piglets after inoculation with PRV-GD2013, PRV-GD2013-ΔgI/gE, or DMEM. b gB-specific antibody levels. Sample with S/N ratios ≤0.60, were classified as positive for gB antibodies. c Detection of gE-specific antibody levels. Samples with S/N ratios ≤0.60 were classified as positive for gE antibodies. All data are presented as the mean ± SD

Fig. 5. a Pathological examination of organ tissues. Groups of piglets (n = 3) were inoculated with 105 TCID50 PRV-GD2013, 105 TCID50 PRV-GD2013-ΔgI/gE, or DMEM. At 17 dpi, all surviving piglets were euthanized and necropsied. Tissue samples from the brain, lymph nodes, lung, kidney, liver, spleen were collected and used for pathological examination. b Pathological changes in various organ tissues of immunized piglets that were challenged with PRV-GD2013. Groups of piglets (n = 5) were inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, 104 TCID50 PRV-GD2013-ΔgI/gE, 103 TCID50 PRV-GD2013-ΔgI/gE, Bartha-K61, or DMEM. At 14 dpc, all surviving piglets were euthanized and necropsied. Tissue samples from the brain, lymph nodes, lung, kidney, liver, spleen were collected and used for pathological examination

Fig. 6. Immunization and challenge experiments in piglets. a Rectal temperature of piglets after challenge with PRV-GD2013. b gB-specific antibody levels. Samples with S/N ratios ≤0.60 were classified as positive for gB antibodies. c gE-specific antibody levels. Samples with S/N ratios ≤0.60 were classified as positive for gE antibodies. All data are presented as the mean ± SD

Fig. 7. Histological examination of brain (A1–A5), lung (B2-B5), liver (C2–C5), spleen (D2–D5), kidney (E2–E5), and lymph nodes (F2–F5) of the piglets in different groups. A1–F1 Correspond to piglets in 105 TCID50 PRV-GD2013-ΔgI/gE vaccinated groups. A2–F2 Correspond to piglets in 104 TCID50 PRV-GD2013-ΔgI/gE vaccinated groups. A3–F3 Correspond to piglets in 103 TCID50 PRV-GD2013-ΔgI/gE vaccinated groups. A4–F4 Correspond to piglets in Bartha k61 vaccinated groups. A5–F5 Correspond to piglets in unvaccinated groups. Histopathologic examination and H&E staining. Magnification, 200 ×