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

Abstract

White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double-stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA-specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei. From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA-ORF89-producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA-ORF89. Yeast colonies positive to the WSSV-ORF89 gene were selected. The expression of dsRNA-ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA-ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA-ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01-AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA-ORF89 able to protect WSSV-challenged shrimp.