Abstract：
: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.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Saksmerprome V,Charoonnart P,Gangnonngiw W,Withyachumnarnkul Bdoi
10.1016/j.jviromet.2009.08.010subject
Has Abstractpub_date
2009-12-01 00:00:00pages
213-7issue
1-2eissn
0166-0934issn
1879-0984pii
S0166-0934(09)00389-9journal_volume
162pub_type
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