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. 2018 Sep 12;9:2071. doi: 10.3389/fimmu.2018.02071

Figure 2.

Figure 2

Antiviral mechanism of mja-miR-35 in shrimp. (A) Predicted target genes of mja-miR-35. As predicted, the 3′ UTRs of wsv140, wsv279, wsv309, and wsv361 genes were targeted by mja-miR-35. The seed sequence of mja-miR-35 was underlined. (B) Constructs of the wild-type and mutated 3′ UTRs of the viral gene. The sequences complementary to the seed sequence of mja-miR-35 or the mutated seed sequences were underlined. (C) Interaction between mja-miR-35 and the viral wsv140, wsv279, wsv309, and wsv361 genes in insect cells. The insect High Five cells were co-transfected with mja-miR-35 and different plasmids (EGFP-3′ UTR or EGFP-3′ UTR-mutant). As a control, mja-miR-35-scrambled was included in the transfection. At 48 h after transfection, fluorescent images (upper panel) and fluorescence intensities (lower panel) were obtained. Lane headings indicated the plasmids used. The miRNAs for transfections were shown on the left. Scale bar, 10 μm. (D) Overexpression of mja-miR-35 in shrimp. Shrimp were co-injected with WSSV and mja-miR-35 or mja-miR-35-scrambled. PBS and WSSV alone were used as controls. At 48 h after injection, shrimp hemocytes were subjected to Northern blotting with mja-miR-35-specific probe. U6 was used as a loading control. (E) Effects of mja-miR-35 overexpression on viral gene expression in vivo. WSSV- infected shrimp were injected with mja-miR-35 or scrambled-miRNA. At 24 h and 48 h after injection, the shrimp hemocytes were subjected to quantitative real-time PCR to examine the expression levels of wsv140, wsv279, wsv309, and wsv361 genes. (F) Silencing of virus gene expression in shrimp. Sequence-specific siRNA was injected into WSSV-infected shrimp to knock down the expression of virus genes. At different time points after injection, the shrimp hemocytes were analyzed using quantitative real-time PCR. WSSV alone and scrambled-siRNA were used as controls. (G) Influence of virus gene silencing on WSSV replication. Gene-specific siRNA or scrambled-siRNA was injected into WSSV-infected shrimp. At different time points after infection, the hemocytes of shrimp were subjected to quantitative real-time PCR analysis to detect WSSV copies. WSSV alone was used as a positive control. (H) Effects of virus gene silencing on the mortality of WSSV-infected shrimp. The shrimp were injected with different siRNAs. The shrimp mortality was monitored daily. WSSV alone was used as a control. In all panels, the statistically significant difference between treatments was indicated with asterisks (*p < 0.05; **p < 0.01).