Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 14;17(1):164.
doi: 10.1186/s12917-021-02861-6.

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

Wenhui Li  1   2 , Dijing Zhuang  1   2 , Hong Li  3 , Mengpo Zhao  1   2 , Erpeng Zhu  1   2 , Baoming Xie  1   2 , Jinding Chen #  1   2 , Mingqiu Zhao #  4   5
Affiliations

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

Wenhui Li et al. BMC Vet Res. .

Abstract

Background: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry.

Results: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013.

Conclusions: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Keywords: PRV-GD2013-ΔgI/gE; Pseudorabies virus; Recombinant virus; Vaccine.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
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
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
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
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
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
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
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 ×
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jons A, Gerdts V, Lange E, Kaden V, Thomas C. Mettenleiter. Attenuation of dUTPase-deficient pseudorabies virus for the natural host. Vet Microbiol. 1997;56(1–2):47–54. doi: 10.1016/S0378-1135(96)01353-3. - DOI - PubMed
    1. Dijkstra JM, Gerdts V, Klupp BG, Mettenleiter TC. Deletion of glycoprotein gM of pseudorabies virus results in attenuation for the natural host. J Gen Virol. 1997;34(9):2147–2151. doi: 10.1099/0022-1317-78-9-2147. - DOI - PubMed
    1. Klupp BG, Lomniczi B, Visser N, Fuchs W, Mettenleiter TC. Mutations affecting the UL21 gene contribute to avirulence of pseudorabies virus vaccine strain Bartha. Virology. 1995;212(2):466–473. doi: 10.1006/viro.1995.1504. - DOI - PubMed
    1. Wittmann G, Rziha HJ. Aujeszky’s disease (Pseudorabies) in pigs. Dev Vet Virol. 1989;9:230–325. doi: 10.1007/978-1-4613-1587-2_7. - DOI
    1. Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of Pseudorabies virus: impact on Neurovirology and veterinary medicine. Microbiol Mol Biol Rev. 2005;69(3):462–500. doi: 10.1128/MMBR.69.3.462-500.2005. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources