Double-stranded RNA-mediated silencing of the white spot syndrome virus VP28 gene in kuruma shrimp, Marsupenaeus japonicus

要旨

Recent applications of RNA interference technology against the white spot syndrome virus (WSSV) were found to be promising and effective antiviral strategies. In the present study, a gene-specific full-length double-stranded RNA (dsRNA) was prepared for the WSSV-VP28 gene using the in vitro transcription method. Challenging experiments were performed by an intramuscular injection of VP28-dsRNA in Marsupenaeus japonicus, followed by WSSV infection after 24 h, expecting 95–100% mortality within in 9 days of duration. The results revealed that the protective efficiency of VP28-dsRNA against WSSV infection is more than 85%, 25 days post infection (dpi). Various assays including bioassay, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry (IHC), histology and 4′,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the knockdown of the VP28 gene. No positive signals were observed in the gill and heart tissues of VP28-dsRNA/WSSV-infected shrimp 7 dpi by RT-PCR and Western blotting. No infection was observed in the VP28-dsRNA/WSSV-infected group through histology and IHC. DAPI staining of haemocytes revealed no signs of condensation and fragmentation in the VP28-dsRNA-administered group. The unrelated YHVGP116-dsRNA had no significant effect on the expression of the VP28 gene, confirming that the knockdown is specific to VP28-dsRNA.

主題