The commercial PRRSV attenuated vaccine can be a potentially effective live trivalent vaccine vector

Figures

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  Fig. 1. Design, construction, and rescue of the PRRSV rGD-EGFP infectious clone. A Strategy used for constructing the full-length PRRSV rGD-EGFP strain. B Cytopathic effect (CPE) and green fluorescence induced by rescued chimeric PRRSV rGD-EGFP in MARC-145 cells at different time points. MARC-145 cells were infected with the supernatants removed from BHK-21 cells transfected by recombinant pBAC-GD-EGFP plasmids. C A Western blotting assay was used to detect the expression of PRRSV-N protein and EGFP protein in MARC-145 cells infected with the PRRSV rGD-EGFP strain. PRRSV rGD was used as a positive control. D The viral titer of the rescued chimeric PRRSV rGD-EGFP was determined in MARC-145 cells. MARC-145 cells were infected with rGD-EGFP or rGD (MOI = 0.1); PRRSV rGD was used as a control

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  Fig. 2. Design, construction, and rescue of the PRRSV rGD-mCherry infectious clone. A Strategy used for constructing the full-length PRRSV rGD-mCherry strain. B CPE and red fluorescence induced by rescued chimeric PRRSV rGD-mCherry in MARC-145 cells at different time points. MARC-145 cells were infected with the supernatants removed from BHK-21 cells transfected by recombinant pBAC-GD-mCherry plasmids. C A Western blotting assay was used to detect the expression of PRRSV-N protein and mCherry protein in MARC-145 cells infected with the PRRSV rGD-mCherry strain. PRRSV rGD was used as a positive control. D The viral titer of the rescued chimeric PRRSV rGD-mCherry was determined in MARC-145 cells. MARC-145 cells were infected with rGD-mCherry or rGD (MOI = 0.1), PRRSV rGD was used as a control

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  Fig. 3. Design, construction, and rescue of the PRRSV rGD-mCherry-EGFP infectious clone. A Strategy used for constructing the full-length PRRSV rGD-mCherry-EGFP strain. B Fluorescence induced by rescued chimeric PRRSV rGD-mCherry in MARC-145 cells at different time points. MARC-145 cells were infected with the supernatants removed from BHK-21 cells transfected by recombinant pBAC-GD-mCherry-EGFP plasmids. C A Western blotting assay was used to detect the expression of PRRSV-N, mCherry, and EGFP proteins in MARC-145 cells infected with the PRRSV rGD-mCherry-EGFP strain. PRRSV rGD was used as a positive control. D The viral titer of the rescued chimeric PRRSV rGD-mCherry-EGFP was determined in MARC-145 cells. MARC-145 cells were infected with rGD-mCherry-EGFP or rGD (MOI = 0.1), PRRSV rGD was used as a control

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  Fig. 4. The stability of EGFP and mCherry expression of the recombinant PRRSV rGD-mCherry-EGFP strain. A Green and red fluorescence were observed in MARC-145 cells infected with the PRRSV rGD-mCherry-EGFP strain of different generations. PRRSV rGD-mCherry-EGFP strains from P1 to P10 generations were used to infect MARC-145 cells, and red and green fluorescence were observed 24 h post infection. Uninfected MARC-145 cells were used as a negative control. B A Western blotting assay was used to detect the expression of PRRSV-N, mCherry, and EGFP proteins in MARC-145 cells infected with PRRSV rGD-mCherry-EGFP strains from P1 to P10 generations. PRRSV rGD was used as a positive control, and the uninfected MARC-145 cells were used as a negative control. C The virus titers of different generations of PRRSV rGD-mCherry-EGFP strains. MARC-145 cells were infected with the PRRSV rGD-mCherry-EGFP strain of different generations at MOI = 0.1, and then the viral titer was determined according to the Reed-Muench method. D The genetic stability of EGFP and mCherry in rGD-mCherry-EGFP-infected MARC-145 cells. Viral genomic RNA was extracted by RT-PCR with primers listed in Table 1, and the products were…

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  Fig. 5. EGFP and mCherry can work as indicators of the infection of the PRRSV rGD-mCherry-EGFP strain. A, B The intracellular and intercellular spread of rGD-mCherry-EGFP-infected MARC-145 cells. MARC-145 cells were infected with the PRRSV rGD-mCherry-EGFP strain with 0.1 MOI, and after 12 h, the localization of green fluorescent protein, red fluorescent protein, and PRRSV N protein in the cells was observed using indirect immunofluorescence assay (IFA). C Flow cytometry is used to detect the number of EGFP-mCherry-positive cells in MARC-145 cells infected with the PRRSV rGD-mCherry-EGFP strain at different hours post infection

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  Fig. 6. rGD-mCherry-EGFP can be used as a convenient tool for antiviral drug screening. A The toxicity of ribavirin on MARC-145 cells at working concentration was determined by CCK-8 assay. B Evaluate the anti-PRRSV infection effect of different concentrations of ribavirin in MARC-145 cells by directly observing green fluorescence and red fluorescence. MARC-145 cells were infected with a mixture of different concentrations of ribavirin and the PRRSV rGD-mCherry-EGFP strain and observed green and red fluorescence after 24 h. C The Western blotting assay is also used to identify the antiviral effect of ribavirin. MARC-145 cells infected only with the PRRSV rGD-mCherry-EGFP strain were used as a positive control, while MARC-145 cells uninfected were used as a negative control

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  Fig. 7. Rectal temperature records and serological response against PRRSV, EGFP, and mCherry after PRRSV rGD-mCherry-EGFP vaccination. A Weekly rectal temperature following challenge with PRRSV rGD-mCherry-EGFP strain. B ELISA (S/P ratio) analysis for humoral immune response against PRRSV N protein, using serum samples from animals in the rGD-mCherry-EGFP-vaccinated group and the mock group, collected at the indicated times. C Western blotting assay analysis for humoral immune response against EGFP and mCherry proteins. Serum samples were collected at the indicated times. D Western blotting assay analysis for humoral immune response against MARC-145 cell samples infected with PRRSV rGD, rGD-EGFP, or rGD-mCherry