Zebrafish U6 promoter driving short-hairpin RNA expression for PmRab7 knockdown to inhibit yellow head virus infection in shrimp hemocytes

Abstract

RNA interference (RNAi) was investigated as potential antiviral strategy to mitigate losses in shrimp aquaculture. With this aim, an effective short-hairpin RNA (shRNA) expressed intracellularly from bacterial vectors incorporating eukaryotic promoters offers an alternative to an injected synthetic small-interfering RNA (siRNA) or long double-stranded RNA (dsRNA). The vector-based RNAi designed to contain a U6 snRNA polymerase III promoter sequence from zebrafish (Danio rerio) for driving shRNA was previously introduced to shrimp cell extract and was able to express the shRNA. Here, four DNA plasmids containing putative zebrafish U6 promoter to drive shRNA against PmRab7-specific sequence were used to transfect primary hemocyte culture. The cells were subsequently infected by Yellow head virus (YHV). As results, when analyzed by RT-PCR at 24 hr post-transfection, Penaeus monodon Rab7 (PmRab7) mRNA transcription was inhibited most significantly by the pshPmRab7-2 construct. YHV replication in primary shrimp hemocyte cultures was shown to be inhibited substantially by this PmRab7 gene-specific hpRNA construct. Transfection of pshPmRab7-2 construct also reduced YHV replication most effectively when analyzed similarly between 24 hr until 72 hr post-infection. These results demonstrate a potential application of DNA-based shRNA construct as effective molecule for antiviral therapy in shrimp.