Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp.

Thitiporn Thammasorn,Pakkakul Sangsuriya,Watcharachai Meemetta,Triwit Rattanarojpong,Sarocha Jitrakorn,Saengchan Senapin,Vanvimon Saksmerprome

doi:10.1186/s12896-015-0226-9

Abstract

BackgroundRNA 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.ResultsFor 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.ConclusionThe 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.

Highlights

RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-strandedRNA targeting the viral gene
A single co-cultivation with Terrific Broth medium resulted in high cell density and Double stranded RNA (dsRNA) yield The effect of culture medium on productivity of E. coli expression system has been studied by varying various media to achieve the optimal production of bioactive compounds [21, 22], indicating the need for optimization of culture conditions when attempting cultivation of new species
Previous work by our group demonstrated the production of dsRNA against shrimp viruses in RNase-deficient E. coli using Luria Bertani (LB) broth medium [18, 19, 23, 24]

Summary

Introduction

RNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. 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.”. White spot syndrome virus (WSSV), a major pathogen with high infectivity and mortality, has been a serious threat for penaeid shrimp aquaculture in the past two decades. WSSV is a large double-stranded DNA virus with the approximate genome size of 300 kbp [1,2,3]. Most of their putative translated gene products have no homology to other proteins from viruses or host cells.

Methods

Results

Conclusion