A novel and inexpensive application of RNAi technology to protect shrimp from viral disease.
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 (approximately 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 (approximately 10-15 g) was injected with 25 microg 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.
Saksmerprome
Vanvimon
V
Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. vanvimon.sak@biotec.or.th
Charoonnart
Patai
P
Gangnonngiw
Warachin
W
Withyachumnarnkul
Boonsirm
B
D000818:Animals; D001709:Biotechnology / Q000191:economics / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D033561:Penaeidae / Q000201:enzymology / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D034622:RNA Interference / Q000276:immunology; D012330:RNA, Double-Stranded / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046828:Roniviridae / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase