A novel and inexpensive application of RNAi technology to protect shrimp from viral disease

作者：Saksmerprome Vanvimon^*; Charoonnart Patai; Gangnonngiw Warachin; Withyachumnarnkul Boonsirm

来源：Journal of Virological Methods, 2009, 162(1-2): 213-217.

DOI：10.1016/j.jviromet.2009.08.010

• Shrimp
• RNAi
• Hairpin
• Antiviral immunity
• Penaeus vannamei
• Yellow head virus (YHV)

Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (similar to 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (> 300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (similar to 10-15 g) was injected with 25 mu g of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.

• 出版日期2009-12