Characteristics of Classical Swine Fever Virus Variants Derived from Live Attenuated GPE− Vaccine Seed

Figures

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  Figure 1. The difference in amino acid sequence between the master seed GPE− and vGPE−. The coding regions of the master seed GPE− (GenBank Accession Number D49533.1) and vGPE− are displayed as boxes divided by each protein gene. Structural proteins and nonstructural proteins are shown in gray and in white, respectively. A total of 10 amino acid substitutions are recognized at positions 330 and 374 in Erns, 761 in E2, 1134 and 1333 in NS2, 2563 in NS4B, 2791 and 2792 in NS5A, and 3571 and 3572 in NS5B. The substitution positions are shown in red on the backbone of vGPE−.

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  Figure 2. SK-L cells electroporated with in vitro-transcribed RNAs. The amino acid substitutions (red) were introduced in the backbone of vGPE−. (a) the viral genome of vGPE−-MS possesses the identical amino acid sequence to the master seed GPE−; (b) three viral genomes of vGPE−/S3571R, vGPE−/A3572P, vGPE−/S3571R; A3572P possess substitutions in NS5B; (c,d) the in vitro-transcribed RNAs derived from the viral genomes above were electroporated into SK-L cells. After 72 h, the cells were heat-fixed and immunostained using the anti-NS3 mAb, 46/1. + depicts that the antibody reacted with the viral NS3 proteins: the success of virus rescue.—depicts that the antibody did not react with the viral NS3: failure of virus rescue. The original magnification was ×200.

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  Figure 3. Amino acid alignment of substitution sites among CSFV strains. The amino acid sequences of 10 substitution sites and their contexts of selected CSFV strains are aligned. The sites of amino acid substitution are highlighted in gray. The amino acid sequences unique to the master seed GPE− are highlighted in red.

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  Figure 4. Effect of amino acid substitutions in NS5B on the viral RNA replication efficiency: (a) schematic diagrams of replicon genomes, coding firefly luciferase. The substituted amino acids are shown in red at positions 3571 and 3572 of NS5B; (b) the in vitro-transcribed replicon RNAs were electroporated into CPK cells. After 3 h, 12 h, 24 h, and 48 h of incubation, the firefly luciferase activity, expressed in relative light units, was assayed in cell lysates. The firefly luciferase activity was normalized with that of the replication-deficient rGPE−-Npro-Luc-IRES-NS3/GAA replicon at the initial time point. Each bar represents the mean value of the triplicates, with error bars showing the standard deviations. The significance of relative luciferase activity differences with replication-competent rGPE−-Npro-Luc-IRES-NS3 or replication-incompetent rGPE−-Npro-Luc-IRES-NS3/GAA was calculated with Student’s t-test. * indicates p < 0.05 compared with the rGPE−-Npro-Luc-IRES-NS3. # indicates p < 0.05 compared with the rGPE−-Npro-Luc-IRES-NS3/GAA.

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  Figure 5. Characteristics of vGPE−, vGPE−/MS8, and vGPE−/MS9 in vitro: (a) the viral genomes of vGPE−/MS8 and vGPE−/MS9 are shown; (b) the in vitro-transcribed RNAs of vGPE−/MS8 and vGPE−/MS9 were electroporated into the SK-L cells. After 72 h, the cells were heat-fixed and immunostained using the anti-NS3 mAb, 46/1. + depicts that the antibody reacted with the viral NS3 proteins: the success of virus rescue.—depicts that the antibody did not react with the viral NS3: failure of virus rescue. The original magnification was ×200; (c,d) the pGPK cells and SK-L cells were infected with vGPE−, vGPE−/MS8, or vGPE−/MS9 at a multiplicity of infection (MOI) of 0.001. The pGPK and SK-L cells were incubated at 30 °C and 40 °C in the presence of 5% CO2, respectively. The supernatants were collected at indicated times. The titers were measured in SK-L cells and expressed as 50% tissue culture infective dose (TCID50) per mL. The IFN-α/β bioactivity in the supernatants of SK-L cells was measured using the SK6-MxLuc cells. Error bars represent the standard deviations. The significance of mutant viral growth differences was calculated using one-way ANOVA followed by Student’s t-test with Bonferroni…