Vanvimon SaksmerpromePatai CharoonnartWarachin GangnonngiwBoonsirm WithyachumnarnkulMahidol UniversityThailand National Center for Genetic Engineering and Biotechnology2018-09-132018-09-132009-01-01Journal of Virological Methods. Vol.162, No.1-2 (2009), 213-217016609342-s2.0-70349734896https://repository.li.mahidol.ac.th/handle/20.500.14594/27746Large-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 (∼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 (∼10-15 g) was injected with 25 μg 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. © 2009 Elsevier B.V. All rights reserved.Mahidol UniversityImmunology and MicrobiologyArticleSCOPUS10.1016/j.jviromet.2009.08.010