PRODUCTION OF TWO SPECIFIC DOUBLE-STRANDED RNAS AGAINST YELLOW HEAD AND WHITE SPOT SYNDROME VIRUSES IN Litopenaeus vannamei

Abstract

Pacific white shrimp (Litopenaeus vannamei) is one of important aquatic animals in Thailand making a high income per year. However, outbreaks of diseases caused by yellow head virus (YHV) and white spot syndrome virus (WSSV) infection result in tremendous economic loss. Nowadays, dsRNA-mediated RNAi becomes the most promising technique to control these viruses. It has been shown that the best target genes for efficient inhibition of YHV and WSSV are protease and ribonuleotide reductase small subunit (rr2), respectively. With the sequence specificity of this RNAi technique, one dsRNA can suppress only one virus. Therefore, production of one molecule of dsRNA (multi-targeted dsRNA) to effectively inhibit both YHV and WSSV is needed. In this study, two types of recombinant plasmid (pET17b-dspro-rr2 and pET17b-dspro-dsrr2) that can produce two different forms of multi-targeted dsRNA (one-stem and two-stem) in E. coli were constructed. The multi-target dsRNA was designed specifically to both protease gene of YHV and rr2 gene of WSSV. After production of dsRNA in HT115 for 4 hours, weight ratio of dsRNA-protease and dsRNA-rr2 from the one-stem was 1:1 while that of two-stem was 1:4. The potency of each multi-targeted dsRNA on viral inhibition and shrimp mortality reduction were investigated in shrimp. Shrimp were injected with one-stem or two-stem into hemolymph before receiving YHV or WSSV. The results showed that one-stem and two-stem could inhibit both viruses (either separate or dual infection) however one-stem was more effective than two-stem when shrimp were infected by WSSV. One-stem could be maintained in shrimp hemolymph at least 5 days without loss of inhibitory effect whereas two-stem lost suppression level against YHV when dsRNA was 5 days pre-injected into shrimp. This study provides an essential information to design and achieve an effective multi-targeted dsRNA for multiple genes silencing in shrimp and reveals the potential anti-YHV and WSSV strategy for further application in the shrimp farm.