Fig. 6

Overexpression of nedd8 upregulates key antiviral genes after SVCV infection and suppresses viral replication in vivo. (A–D) Ectopic expression of nedd8, induced by mRNA injection upregulated key antiviral genes in SVCV infected zebrafish larvae. We injected zebrafish embryos at the one-cell stage with either GFP mRNA (400 pg/per embryo) or Myc-tagged nedd8 mRNA (400 pg/per embryo). At 3 dpf, we added SVCV viruses (2 × 10⁸ TCID₅₀/ml) into the water containing zebrafish larvae. After incubation for 24 h, we extracted total RNA from all larvae and performed qPCR assays to detect the expression levels of ifn1 (A), mxc (B), and pkz (C) and lta (D). (E–G) Ectopic expression of nedd8 by mRNA injection suppressed SVCV replication in embryos. We performed qRT-PCR assays to detect the expression levels of the SVCV genes P (E), G (F), and N (G) genes of SVCV. (H,I) Western blot assay and Fluorescence micrographs of zebrafish embryos showed the expression levels of injected GFP mRNA or Myc-nedd8 mRNA. Data are shown as mean ± SEM of three independent experiments, each performed in triplicate; the statistical analysis was performed using GraphPad Prism 5 (unpaired t-test).