Figure 5

Antiviral function of Bmp4 in vivo. (A) The generation of bmp4 mutation zebrafish using CRISPR/Cas9 technology. a. The knockout gene target is located in exon 3. The black box represents exons. The fold line represents introns. The white square represents un-translated regions. b and c. Compared with the wild type, 1 nucleotide was replaced by 11 nucleotides in exon 3 of bmp4 in the mutant, leading to early termination of translation to the 118th amino acid. (B) Kaplan-Meier ana-ysis of the overall survival of WT (n = 30) or bmp4^−/− zebrafish larvae (n = 30), which were infected by adding GCRV into the embryo culture medium (final 6 × 10⁶ TCID₅₀/mL) and monitored every 6 h after infection. (C) The symptoms of adult zebrafish that were infected with the virus of GCRV. (D) Kaplan-Meier analysis of the overall survival of WT (n = 30) or bmp4^−/− zebrafish (n = 30) that were injected i.p. with 40 µL of GCRV (2.5 × 10⁷ TCID₅₀/mL) and monitored every day after infection. (E,F) The expression of GCRV RNA in the liver and spleen from wild-type (WT) or bmp4^−/− zebrafish injected i.p. with 40 µL of GCRV (2.5 × 10⁷ TCID₅₀/mL). The expression of zebrafish actb1 was used as an internal control for the qRT-PCR. Data were from three independent experiments. Data were analyzed using Student’s t-test (two-tailed) and are presented as mean ± SD (*** p < 0.001).