Foot-and-mouth disease virus vaccine with VP1 G-H loop substitution of the Cathay strain broadens antigen spectrum

Figures

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  Fig. 1. Schematic diagram of the exchange-cassette cloning strategy described in the present study. The FMDV full-length cDNA clone of pOFS was used for the generation of a chimeric genome-length plasmid of pOFS/XJLP1, which contained the substitution of leader protein (L) and structural protein (P1) of O/XJ/CHA/2017. Based on this construct, three recombinant full-length cDNA clones were generated by substituting the VP1 G-H loop (130–160 aa) of different FMDVs. UTR, untranslated region

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  Fig. 2. Analysis of the recombinant FMDVs by IFA. Seventy to 80% confluent BHK-21 cells were infected with the parental virus or the recombinant FMDVs. After 6–8 h incubation at 37 °C, the monolayer cells were fixed and stained by monoclonal antibody against FMDV 3 A and Texas red-conjugated secondary antibody. The cells were observed under a laser-scanning confocal microscope. Scale bars, 5 µm

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  Fig. 3. The CPE in BHK-21 cells infected with the recombinant FMDVs and the parental virus of p6 was observed at 12 hpi. A Control BHK-21 cell; B CPE in BHK-21 cells infected with the parental virus; CPE score is “ ± ”; C–E CPE in BHK-21 cells infected with the mutant viruses of rHN/XJ/TURG-H, rHN/XJ/HBG-H and rHN/XJ/TURG-H; CPE scores all are “ + + + + ”

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  Fig. 4. The plaque phenotypes and one-step growth curves of the parental virus and the mutant FMDVs on BHK-21 monolayers. A BHK-21 cells were infected with the indicated dilutions of the parental virus and the recombinant FMDVs, and monolayers were stained with crystal violet at 48 hpi. B BHK-21 cell monolayers were infected with the parental virus and the mutant FMDVs at a MOI of 5. At the times indicated, the supernatants were harvested, and the virus titers were detected by plaque assay on BHK-21 cells. The viral titers were obtained from triplicate experiments

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  Fig. 5. FMDV‑specific antibody production in vaccinated pigs. Groups of five pigs were vaccinated with the mutant virus vaccines and the parental virus vaccine. Blood samples were collected on days 0, 7, 14, 21, and 28 dpv and assayed by LPBE ELISA. The results represent the mean value of five animals. Error bars indicate SDs from the mean

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  Fig. 6. Mean neutralizing antibody titers against homologous and heterologous FMDVs in pigs. Groups of five pigs were respectively inoculated with inactivated vaccines prepared from the mutant viruses and the parental virus. Sera collected from vaccinated pigs at 28 dpv were used to detect virus neutralizing antibodies against type O FMDVs belonging to four different lineages. Median values are indicated with error bars. Statistical analysis was conducted using an unpaired t test of GraphPad Prism Software (version 8.3.0) (*p < 0.05, **p < 0.01, ***p < 0.001). The dotted line indicates the cut-off level of virus-neutralizing titer of 1.65 Log10

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  Fig. 7. Mean neutralizing antibody titers against FMDV O/GDLeiZh/2020 in pigs. Sera collected from pigs vaccinated with four experimental vaccines at 28 dpv were used to detect virus neutralizing antibodies against FMDV O/GDLeiZh/2020. Median values are indicated with error bars. Statistical analysis was conducted using an unpaired t test (*p < 0.05, **p < 0.01, ***p < 0.001) of GraphPad Prism Software (version 8.3.0). The dotted line indicates the cut-off level of virus-neutralizing titer of 1.65 Log10

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  Fig. 8. Antigenic relationship (r1) values of four mutant FMDVs and the field viruses. A two-dimensional (2D) virus neutralization test was performed using sera from five pigs vaccinated with the four experimental vaccines at 28 dpv. The horizontal dotted line indicates the cut-off value of 0.3. The mean r1 values were obtained from three replicates of five animals. Error bars indicate SDs from the mean

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  Fig. 9. Alignment of the VP1 amino acid sequences. Amino acid sequences of the VP1 of O/Tibet/CHA/99, O/TUR/5/2009, O/XJ/CHA/2017, O/HBCZ/2019, O/18074, and O/GDLeiZh/2020 were aligned using MEGA 6.06. Dots represent amino acid identities with O/Tibet/CHA/99