Effects of length and sequence of long double-stranded RNAs targeting ribonucleotide reductase 2 of white spot syndrome virus (WSSV) on protective efficacy against WSSV

Abstract

Long double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has been a well-known mechanism against white spot syndrome virus (WSSV) in cultured shrimp. In the present study, we investigated the protective efficacy of dsRNAs targeting the ribonucleotide reductase 2 (rr2) gene of WSSV according to length and target sequence location. To produce different lengths of dsRNAs, the 640 bp rr2 fragment (fragment I) was split into two equal 320 bp fragments (fragment II and III), then each 320 bp fragment was redivided into two 160 bp fragments (fragment IV, V, VI, and VII). After the synthesis of seven kinds of dsRNA fragments, dsRNAs with the same length were mixed with each other, then used for the evaluation of dsRNA's length effect in Penaeus vannamei. The result showed that 160 bp long dsRNAs were as effective as 320 and 640 bp long dsRNAs in the protection of shrimp against WSSV infection, suggesting that the dsRNA length of 160 bp would be enough to be used as RNAi-mediated WSSV suppression in P. vannamei. However, as the 160 bp long dsRNAs used in the length effect experiment were not a single dsRNA population but a mixture of 160 bp dsRNA fragments covering the parent 640 bp long dsRNA, the sequence effect was not included in this RNAi efficacy. In the experiments to know the effect of not only length but also sequence of rr2-targeting long dsRNAs on the protective efficacy against WSSV, dsRNAs with a length of 640 bp (fragment I) and 320 bp (fragment II, III) showed a constant high defense ability, but the protection degree of long dsRNAs with a length of 160 bp was different depending on the kinds of the fragment, suggesting that the RNAi efficacy of some rr2-targeting long dsRNAs with a length of 160 bp might have sequences that are variable according to experimental conditions. In conclusion, this study showed that the protective ability of long dsRNAs in shrimp against WSSV infection can be affected by the length and sequence of the long dsRNAs.

Keywords: Length of dsRNA; Penaeus vannamei; RNA interference; Ribonucleotide reductase 2; Sequence of dsRNA; WSSV.