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

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.

RESPONS IMUN UDANG WINDU Penaeus monodon TERHADAP VAKSIN dsRNA VP-24 PADA DOSIS BERBEDA

“…Viral protein ini berpeluang untuk dijadikan sebagai vaksin rekombinan (Sánchez-Paz, 2010). Teknologi interferensi RNA (RNAi) merupakan teknik vaksinasi DNA rekombinan yang spesifik dan efektif untuk menghambat replikasi virus dengan introduksi small interference (si)RNA maupun double-stranded (ds)RNA pada target gen virus (Haq et al, 2012;Jayachandran et al, 2012;Thammasorn et al, 2015). Teknologi vaksinasi menggunakan agen virus dsRNA berkembang sangat pesat terutama dibidang medis, pertanian, dan akuakultur (Jin et al, 2010;Lichner et al, 2003;Rowley & Pope, 2012).…”

Production of specific dsRNA against white spot syndrome virus in the yeastYarrowia lipolytica

“…On the other hand, Thammasorn et al. () orally administered dsRNA‐vp28 (12 μg/g food) to 3–5 g L. vannamei shrimp and challenged them with WSSV, finding 70% of protective effect. Finally, with the specific dsRNA expression system obtained from the microalgae Chlamydomonas reinhardtii , 13% of protective was obtained using 10 8 dsRNA‐RdRP‐expressing microalgae/gr food in 0.1‐g L. vannamei shrimp.…”

RNA interference in shrimp and potential applications in aquaculture

“…In fact, researchers in Thailand recently succeeded in using RNAi by oral delivery against WSSV infection in P. monodon , leading to reduced percentages in cumulative mortality and delayed average time of death (Thammasorn et al . ).…”

“…While the studies on the applicability of RNAi to control the virus were mostly conducted on a small scale, the success of these experiments clearly showed that RNAi has enormous potential in inhibiting the replication of the virus and improve the survival of shrimp. In fact, researchers in Thailand recently succeeded in using RNAi by oral delivery against WSSV infection in P. monodon, leading to reduced percentages in cumulative mortality and delayed average time of death (Thammasorn et al 2015).…”