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.

中文翻译：

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斑马鱼 U6 启动子驱动 PmRab7 敲低的短发夹 RNA 表达以抑制虾血细胞中的黄头病毒感染

RNA 干扰 (RNAi) 被研究作为潜在的抗病毒策略来减轻对虾养殖中的损失。出于这个目的，一种有效的短发夹 RNA (shRNA) 从结合真核启动子的细菌载体细胞内表达，为注射的合成小干扰 RNA (siRNA) 或长双链 RNA (dsRNA) 提供了替代方案。基于载体的 RNAi 设计包含来自斑马鱼 ( Danio rerio ) 的 U6 snRNA 聚合酶 III 启动子序列) 用于驱动 shRNA 之前被引入虾细胞提取物中，并且能够表达 shRNA。在这里，四个含有假定斑马鱼 U6 启动子的 DNA 质粒用于驱动针对 PmRab7 特定序列的 shRNA 用于转染原代血细胞培养物。这些细胞随后被黄头病毒 (YHV) 感染。结果，在转染后 24 小时通过 RT-PCR 分析时，斑节对虾Rab7 (PmRab7) mRNA 转录受到pshPmRab7-2 构建体的最显着抑制。这种 PmRab7 基因特异性 hpRNA 构建体显着抑制了原代虾血细胞培养物中的 YHV 复制。在感染后 24 小时至 72 小时之间进行类似分析时，pshPmRab7-2 构建体的转染也最有效地减少了 YHV 复制。这些结果证明了基于 DNA 的 shRNA 构建体作为虾抗病毒治疗的有效分子的潜在应用。