Development of a Genetically Engineered Bivalent Vaccine against Porcine Epidemic Diarrhea Virus and Porcine Rotavirus

Figures

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  Figure 1. Preparation of monoclonal antibody against PoRV VP7. (A) Schematic diagram of the recombinant plasmid expressing PoRV VP7. (B) The SDS-PAGE analysis of the rHis-VP7 protein that was eluted by different concentrations of imidazole elution. (C) HEK-293T cells were transfected with the PoRV-VP7 expression plasmid. At 24 h post-transfection, VP7 expression was detected by IFA using mAb 3H5 (magnification, ×100). (D) MA104 cells were infected with 0.1 MOI PoRV. After 18 h post-infection, VP7 expression in PoRV-infected cells was examined by IFA using mAb 3H5 (magnification, ×100). (E) PoRV-infected MA104 cells was analyzed at 18 h post-inoculation by Western blotting using the anti-VP7 mAb 3H5.

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  Figure 2. Rescue of rPEDV-PoRV-VP7. (A) Schematic representation of the cDNA clone of rPEDV-PoRV-VP7. (B) The CPE of rescued rPEDV-PoRV-VP7 in Vero cells was observed by microscope (magnification, ×100). (C) Identification of the ORF3 replacement with PoRV VP7 by whole-genome sequencing.

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  Figure 3. Characteristics of rPEDV-PoRV-VP7. (A) PoRV VP7 and PEDV S were detected in rPEDV-PoRV-VP7-, wild-type PEDV-, or mock-infected Vero cells by IFA (magnification, ×100), respectively. (B) Representative plaques of rPEDV-PoRV-VP7- or wild-type PEDV-infected Vero cells at 20 hpi. (C) Vero cells were infected with rPEDV-PoRV-VP7 or wild-type PEDV at an MOI of 0.01, and cell cultures were collected at indicated times to determine the viral titers by TCID50.

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  Figure 4. The weekly weight gain of rPEDV-PoRV-VP7-vaccinated piglets. Average weekly weight gain values are shown as the means ± SD. N.S, no significance.

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  Figure 5. IgA levels in the rYN150-PoRV-VP7-vaccinated piglets. IgA antibodies against PoRV VP7 (A) and PEDV S1 (B) in oral swabs were collected from the rYN150-PoRV-VP7-inoculated piglets at 0, 7, 14, 21, and 28 DPI were measured by indirect ELISA using His-VP7 or S1-mFc. Mean ± SDs from five piglets inoculated or mock-inoculated with rYN150-PoRV-VP7. ** p < 0.01, as compared with the mock-inoculated group.

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  Figure 6. The titers of IgG and neutralizing antibody in serum from the rPEDV-PoRV-VP7-inoculated piglet. Levels of the anti-PoRV VP7-specific (A) and anti-PEDV S1-specific (B) IgG antibody. Neutralizing activity against PoRV (C) and PEDV (D) in serum was examined by viral neutralization assay in Vero cells. Each point represents the titer of an individual piglet. Data are shown as mean ± SD. * p < 0.05 and ** p < 0.01, as compared with the mock-inoculated group.