Construction of eGFP-Tagged Senecavirus A for Facilitating Virus Neutralization Test and Antiviral Assay

Figures

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  Figure 1. Construction of rSVA-eGFP cDNA clone for rescuing eGFP-expressing recombinant SVA. Schematic representation of rSVA-eGFP cDNA clone (A). The eGFP-T2A fusion sequence is inserted between the 2A and 2B genes in the backbone of rSVA cDNA clone, flanked by the T7 promoter and the Not I recognition sequences at its 5′ and 3′ ends, respectively. Two pairs of primers, F1/R1 and F2/R2 (black arrow-marked), are designed for RT-PCR analyses on rSVA-eGFP genome and on foreign sequence, respectively. Schematic representation of two self-cleavage sites (red arrow-marked) in the rSVA-eGFP polyprotein (B). Agarose gel electrophoresis of Not I-linearized rSVA-eGFP cDNA clone (C). The rSVA-eGFP cDNA clone-transfected BSR cell monolayers at 24, 48 and 72 hpt (D). BSR cell monolayers infected with the rescued rSVA-eGFP at P1, P2, P3 and P4 at 24 hpi (E). BF: bright field (the same as following figures).

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  Figure 2. Identification and characterization of rescued rSVA-eGFP. BSR cell monolayer infected with P5 rSVA-eGFP at 24 hpi (A). RT-PCR analysis of rSVA-eGFP using the F1/R1 primer pair (B). Indirect immunofluorescence assay of rSVA-eGFP-infected BSR cells using the anti-VP1 MAb (primary antibody) and the Alexa Fluor® 555 conjugate (secondary antibody) (C). Observation of ultrathin section of rSVA-eGFP-infected BSR cell by transmission electron microscope (D). BSR cell monolayers infected with rSVA-eGFP at 5, 6, 7, 8, 9 and 10 hpi (E). Fluorescent cytopathic effects (CPE) on cell monolayer infected with rSVA-eGFP at 72 hpi (F). Plaque morphology of the rSVA-eGFP (yellow arrowhead-marked), compared with that of the rSVA (red arrowhead-marked) (G). Growth curve of the rSVA-eGFP, compared with that of the rSVA (H). The data represent the mean ± SD for three independent experiments.

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  Figure 3. Stability of eGFP expressed by the rSVA-eGFP during serial passaging in BSR cells. Cell monolayers infected with rSVA-eGFP at P2, P4, P6, P8, P10, P12, P14 and P16 at 24 hpi (A). RT-PCR analyses of rSVA-eGFP at P6, P8, P10, P12, P14 and P16 using the F1/R1 (B) and the F2/R2 (C) primer pairs, respectively. Brightness of 1004-bp-specific band (C, yellow arrowhead-marked) is gradually decreasing from the P6 to P10, and finally unobservable from the P12 to P16, whereas another band (C, red arrowhead-marked) instead gradually becomes bright from the P12 to P16. The ORF sequence of eGFP, from which a 597-bp sequence (D, black letters) is deleted from the P12 to P16, and which accordingly retains a 120-bp fragment (D, red letters). Sequencing chromatogram of the P16 rSVA-eGFP (E). The black arrow illustrates a possible site, where the 597-bp sequence (D, black letters) is deleted.

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  Figure 4. Development of rSVA-eGFP-based virus neutralization test (VNT) for analysis of serum samples. Schematic representation of 96-well plate format designed for performing rSVA-eGFP-based VNT (A), which shows a hypothetical result that the neutralization titer is determined to be 80 for a given serum sample. The neutralization titer is the dilution of serum that neutralizes rSVA-eGFP in half the wells of a given column. Green and white wells represent fluorescence-emitting and -free cell monolayers, respectively. C.: Control. VNT result of eight porcine serum samples (B), showing that S1, S2, S3 and S4 are neutralization titer-positive; S5, S6, S7 and S8 have no neutralizing activity.

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  Figure 5. Anti-rSVA-eGFP assay of ribavirin in vitro. Cell monolayers with treatment of ribavirin at different concentrations (0, 4, 8, 16, 32 and 64 μM) at 24 hpi (A). Each cell monolayer is observed in a randomly selected field-of-view of fluorescent microscope. Relative viability of BSR cells at 24 h after ribavirin treatment at different concentrations (0, 8, 16, 32, 64, 128, 256, 512, 1024 and 2048 μM) by MTT assay (B). The data represent the mean ± SD for five replicates per dilution. The CC50 value for ribavirin, calculated by nonlinear regression fitting using the GraphPad Prism software (C). Variation in rSVA-eGFP titers at 24 h after ribavirin treatment at different concentrations (0, 4, 8, 16, 32 and 64 μM) (D). The data represent the mean ± SD for three independent experiments. *p < 0.05, **p < 0.01 or ***p < 0.001: comparison between the 0 μM group and another one by two-tailed Student′s t-test. The data in (D) are processed by nonlinear regression fitting using the GraphPad Prism software, and then shown in (E). Variation in relative titers at 24 h after ribavirin treatment at different concentrations (0, 4, 8, 16, 32 and 64 μM) (F). The data represent the mean ± SD for three…

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  Figure 6. Anti-rSVA-eGFP assay of apigenin in vitro. Apigenin- and DMSO-treated cell monolayers at different concentrations at 24 hpi (A). Each cell monolayer is observed in a randomly selected field-of-view of fluorescent microscope. D: DMSO control. Relative viability of BSR cells at 24 h after either apigenin (B) or DMSO (D) treatment at different concentrations by MTT assay. The data represent the mean ± SD for five replicates per dilution. The CC50 value either for apigenin (C) or for DMSO (E), calculated by nonlinear regression fitting using the GraphPad Prism software. Variations in rSVA-eGFP titers at 24 h after either apigenin or DMSO treatment at different concentrations (F). The data represent the mean ± SD for three independent experiments. The data in (F) are processed by nonlinear regression fitting using the GraphPad Prism software, and then shown in (G).