title: Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp.
abstract: BACKGROUND
  RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called "multi-WSSV dsRNA." A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.
  
  
  RESULTS
  For a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.
  
  
  CONCLUSIONS
  The present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. sungsoori@hotmail.com.
authors.forename: Thitiporn
authors.initials: T
authors.lastname: Thammasorn
authors.affiliation: Department of Biochemistry, Center of Excellence for Molecular Biology and Genomics of Shrimp, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. sangsuriya.pk@gmail.com.|National Center of Genetic Engineering and Biotechnology, (BIOTEC), Thailand Science Park, Pathum Thani, 12120, Thailand. sangsuriya.pk@gmail.com.
authors.forename: Pakkakul
authors.initials: P
authors.lastname: Sangsuriya
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. w.meemetta@gmail.com.
authors.forename: Watcharachai
authors.initials: W
authors.lastname: Meemetta
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. saengchan@biotec.or.th.|National Center of Genetic Engineering and Biotechnology, (BIOTEC), Thailand Science Park, Pathum Thani, 12120, Thailand. saengchan@biotec.or.th.
authors.forename: Saengchan
authors.initials: S
authors.lastname: Senapin
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. sarocha.jit@biotech.or.th.|National Center of Genetic Engineering and Biotechnology, (BIOTEC), Thailand Science Park, Pathum Thani, 12120, Thailand. sarocha.jit@biotech.or.th.
authors.forename: Sarocha
authors.initials: S
authors.lastname: Jitrakorn
authors.affiliation: Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand. triwit.rat@kmutt.ac.th.
authors.forename: Triwit
authors.initials: T
authors.lastname: Rattanarojpong
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. vanvimon.sak@biotec.or.th.|National Center of Genetic Engineering and Biotechnology, (BIOTEC), Thailand Science Park, Pathum Thani, 12120, Thailand. vanvimon.sak@biotec.or.th.
authors.forename: Vanvimon
authors.initials: V
authors.lastname: Saksmerprome
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D017756:Aquaculture / Q000379:methods*; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005285:Fermentation; D033561:Penaeidae / Q000276:immunology* / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D046848:White spot syndrome virus 1 / Q000187:drug effects*
chemical_list: D003470:Culture Media; D017931:DNA Primers; D012330:RNA, Double-Stranded