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

• DOI: 10.1111/j.1365-2109.2010.02703.x
• Publication Date: 2010-11-12T04:12:36Z
• Authors: Raja Sudhakaran; Tohru Mekata; Tomoya Kono; Mari Inada; Shogo Okugawa; Maki Yoshimine; Terutoyo Yoshida; Masahiro Sakai; Toshiaki Itami
• Keywords: 0301 basic medicine; 0303 health sciences; 03 medical and health sciences; 3. Good health

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.