A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protects Pigs against PCV2b Challenge and Induces Serum Neutralizing Antibody Response against CSFV

Figures

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  Figure 1. Schematic showing genomic configuration of pseudorabies virus (PRV) and the strategy of thymidine kinase (TK), glycoprotein G (gG), and gE gene deletion to generate a PRV triple mutant virus (tmv) vaccine vector. (A) Schematic of PRV wild type (wt) Becker strain backbone [39]. UL—Unique long region; US—Unique short region; IR—Internal repeat region; TR—Terminal repeat region. Plasmid construct pPRV gEΔ (C) was used to introduce gEΔ in the PRV wt backbone (A) in the gE locus to generate PRV gEΔ mutant virus. Plasmid construct pPRV TKΔ (D) was used to incorporate TK deletion in the PRV gEΔ mutant backbone virus in TK locus (B) to generate PRV gE/TK dual gene deleted mutant virus. Finally, PRVtmv (gE/TK/gG-deleted) was constructed by incorporating the plasmid construct pPRV gGΔ (E) in to the gG locus in PRV gE/TK dual gene deleted mutant virus backbone. Arrows indicate the direction of the corresponding open reading frame (ORF). Nucleotide numbers correspond to the GenBank accession # JF797219. Part.—partial sequence; Inter.—intergenic sequence.

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  Figure 2. Strategy of chimeric PCV2 cap, CSFV Erns-GM-CSF, and CSFV E2 insertion in the TK deletion, gG deletion, and gE deletion loci, respectively, of PRVtmv genome to generate PRVtmv-CSFV E2-Erns-GM-CSF-PCV2b Cap (PRVtmv+). (A) Schematic of PRV triple mutant virus (tmv) genomic organization showing thymidine kinase (TK) (B), glycoprotein G (gG), and gE (C) deletions. Arrows indicate the direction of the corresponding open reading frame (ORF). Nucleotide numbers correspond the GenBank accession # JF797219. (E) Schematic of chimeric PCV2 cap V5 epitope gene expression cassette. The PCV2 cap expression cassette cloned in to EcoRI-NsiI site of pPRV TKΔ to yield pPRV TKΔ/PCV2 Cap-INS. (F) Schematic of chimeric CSFV Erns-GM-CSF Flag expression cassette. The CSFV Erns-GM-CSF expression cassette cloned in to KpnI-BamHI site of pPRV gGΔ to yield pPRV gGΔ/CSFV Erns-GM-CSF-INS (D) Schematic of chimeric CSFV E2 V5 epitope gene expression cassette. The CSFV E2 expression cassette was cloned in to KpnI-BamHI site of pPRV gEΔ to yield pPRVgEΔ/CSFV E2-INS. Part.—partial sequence; Inter.—intergenic sequence.

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  Figure 3. Vaccination, sample collection, challenge, and euthanasia scheme for the animal experiment. Intranasal (IN)—intranasal inoculation; Subcut—subcutaneous injection; PFU—plaque-forming units.

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  Figure 4. Immunoblot analysis of PRVtmv+ expressing chimeric CSFV E2, CSFV Erns-GM-CSF, and PCV2 cap proteins using an anti-CSFV E2 monoclonal antibody (mAbs) (left panel), an anti-CSFV Erns mAbs (middle panel), and a rabbit anti-PCV2 cap Ab (right panel), respectively.

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  Figure 5. Transmission electron microscopy: (A,B) Mock infected healthy swine kidney (SK) cell with normal cellular morphology. (C,D) SK cells were infected with porcine circovirus type 2b (PCV2b) (infected at an MOI of 0.1) and fixed at 72 h post-infection (hpi). PCV2b infected cell showed accumulation of PCV2 viruses (red arrow) within the vesicle-like structures in the cytoplasm. Each PCV2 particle measured about 20 nm in diameter. (E–G) SK cells were infected with PRV wt (infected with an MOI of 5) and fixed at 12 hpi. PRV wt-infected cells showed enveloped herpesvirus (red arrow; about 200 nm in diameter) within the vesicle-like structure in the cytoplasm. The process of budding and release of several enveloped viruses were also noticed on the periphery of the cell near the plasma membrane (blue arrow). Released virus particles from the outer surface of the cells were accumulated in intercellular space (green arrow). (B, D, F, G) are magnifications of (A, C, E), respectively. N—Nucleus; C—Cytoplasm; RER—Rough endoplasmic reticulum; M—Mitochondria; L—Lysosomes.

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  Figure 6. Transmission electron microscopy. (A) PRVtmv+ vaccine virus-infected swine kidney (SK) cells (infected at an MOI of 5) were fixed at 18 h post-infection. PRVtmv+ infected cells showed several fully enveloped 200 nm in diameter size PRVtmv+ vaccine virus particles in the exocytic vesicles in the cytoplasm (red arrows). Secondary envelopment of intracytoplasmic PRVtmv+ capsids by budding into vesicles was visible (yellow arrows). Most of the PRVtmv+ infected SK cells showed several accumulations of PCV2 virus-like particles (VLPs) within the vesicular structures in the cytoplasm (red arrow with a white fill). The PCV2-VLPs were circular, and each measured about 20 nm in diameter. (B) Magnification of A is given. (C) The presence of enveloped PRVtmv+ vaccine virus (red arrow) and PCV2 VLPs within vesicles (red arrow with a white fill) in the cytoplasm of the PRVtmv+ infected SK cells were visible. (D–F) Accumulations of PCV2-VLPs within the vesicle-like structures in the cytoplasm of the infected cells (red arrow with a white fill). C—Cytoplasm; RER—Rough endoplasmic reticulum; G—Golgi apparatus.

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  Figure 7. In vitro characterization of PRVtmv+. (A) Plaque size analysis of PRVtmv+ compared to that of PRV wt. Shown are the pictures of areas containing representative plaques of each virus. (B) Bar graph showing normalized average plaque size (n = 150) for each virus with standard deviation (SD). (C) One-step growth analysis of PRVtmv+ compared with PRV wt.

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  Figure 8. Clinical assessment. (A) Rectal temperature of pigs following immunization and challenge. Rectal temperature was measured using a digital thermometer on indicated days. Shown is the mean temperature of each treatment group with standard deviation (SD) (n = 5). (B) Body weight (BW) of pigs following immunization and challenge. BW was measured using a digital weight balance on indicated days. The mean BW of each treatment group with SD (n = 5) is shown. dpv—days post-vaccination; dpc—days post-challenge. Two-way ANOVA followed by Bonferroni post-tests to compare replicate means by row. p < 0.05 is considered as significant.

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  Figure 9. Nasal shedding of PRVtmv+ in immunized pigs assessed by qPCR and virus isolation. (A) DNA was isolated from nasal swab following immunization with PRVtmv+ vaccine, and PRV-qPCR was performed. PRV genome copy numbers were calculated according to the CT values of a standard curve. Shown are the mean copy numbers of PRV genome in 100 ng of DNA of two independent qPCR analyses of each animal from three vaccination groups on 0, 2, 4, 8, and 15 dpv. The dot plot graph represents mean + individual values in each group (n = 5). (B) Virus isolated from each animal’s nasal swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay. Shown are the virus titers (in plaque-forming unit/mL of the nasal swab; PFU/mL) of each animal from three vaccination groups on 0, 2, 4, 8, and 15 dpv. The dot plot graph represents mean + individual values in each group (n = 5). dpv—days post-vaccination.

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  Figure 10. Quantification of PRVtmv+ in tonsil swab of immunized pigs assessed by qPCR and virus isolation. (A) DNA was isolated from tonsil swabs following immunization with PRVtmv+ vaccine, and PRV-qPCR was performed. PRV genome copy numbers were calculated according to the CT values of a standard curve. The mean copy numbers of PRV genome in 100 ng of DNA from tonsil swab of two independent qPCR analyses of each animal from three vaccination groups on 0, 2, 4, 8, and 15 dpv are shown. The dot plot graph represents mean + individual values in each group (n = 5). (B) Virus isolated from each animal’s tonsil swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay. Shown are the titers (in plaque-forming unit/mL of the nasal swab; PFU/mL) of each animal from three vaccination groups on 0, 2, 4, 8, and 15 dpv. The dot plot graph represents mean + individual values in each group (n = 5).

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  Figure 11. PRVtmv+ is stable in pigs. Immunoblot analysis of PRVtmv+ vaccine virus after a passage in pigs expressing chimeric CSFV E2 and Erns proteins by using E2- (left panel) and Erns-specific (right panel) mAbs, respectively.

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  Figure 12. Indirect immunofluorescence assay (IIFA) for serum samples collected from PRVtmv+ immunized pigs. Swine kidney cells were transfected with PCV2b plasmid. At 72 h post-transfection, cells were fixed and IIFA were performed using serum samples collected from PRVtmv+ immunized pig on 0 day post-vaccination (dpv) (pig #2313) and 32 dpv (pig #2313 and 2304) as a primary antibody and fluorescent-labeled anti-porcine secondary antibody with DAPI nuclear stain. Positive signals were indicated by bright apple-green fluorescent signals. (Magnifications 200×).