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Figure 3 | Veterinary Research

Figure 3

From: CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Figure 3

Biological characteristics of the recombinant virus rPRV HN1201-EGFP-Luc. A One-step growth curves of PRV HN1201 (MOI = 0.1) and rPRV HN1201-EGFP-Luc (MOI = 0.1) in PK-15 cells. This experiment was performed three times, and the results are shown as the mean ± SD. B Percentage survival of mice (eight mice per group) infected I.M. with 106.5 TCID50 and 102.5 TCID50 of PRV HN1201 and rPRV HN1201-EGFP-Luc. C, D Equal numbers of 3D4/21 cells were infected with PRV HN1201 or rPRV HN1201-EGFP-Luc at an MOI of 1. Total RNA was collected at 6, 12 and 24 hpi, and then, IL-1β (C) and IFN-β (D) mRNA expression was quantified by RT-qPCR. This experiment was performed three times, and the results are shown as the mean ± SD. E The rPRV HN1201-EGFP-Luc passage experiments were performed in PK-15 cells (passages 1–20), and then, viral titre was determined with TCID50 assays at specific passages (1, 5, 10, 15 and 20) of rPRV HN1201-EGFP-Luc. Data are shown as the mean ± SD from three independent experiments. F PK-15 cells were infected with specific passages (1, 5, 10, 15 or 20) of rPRV HN1201-EGFP-Luc (MOI = 0.1). Luciferase assays were performed in triplicate at the indicated times after infection. Data are shown as the mean ± SD from three independent experiments. G The plaque assay standardized on genome copy numbers. The number of rPRV HN1201-EGFP-Luc plaques was normalized to the number of PRV HN1201 plaques to detect the effect of gI/gE gene knockout on PRV packaging. Representative data from triplicate experiments are shown. Mean + SD, ns, no significant difference. H Plaque sizes of PK-15 cells infected with PRV HN1201 or rPRV HN1201-EGFP-Luc at 36 hpi. The mean plaque diameter of 50 plaques from one representative experiment out of three independent experiments is shown. Mean + SD, *** P < 0.001.

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