RNA interference mediated gene silencing of essential pathogen genes has been demonstrated to be an effective means to control viral replication in shrimp both by intramuscular injection of targeted dsRNA molecules and by feeding formaldehyde-inactivated E. coli expressing dsRNA. The challenge, however, has been how to continuously produce and deliver RNA interference molecules in shrimp for pathogen control. Pebble Labs has developed a platform technology (Directed Biotics) for continuous production and delivery of RNA interference molecules using engineered bacteria that are naturally associated with the host. Previously, we have demonstrated that feeding shrimp an RNase III-deficient Bacillus subtilis strain expressing dsRNA targeting essential white spot syndrome virus (WSSV) genes was able to reduce shrimp mortality by two-fold in WSSV challenged adult shrimp. To determine which B.subtilis strains were most effective in controlling viral replication by delivery of RNA interference (RNAi) molecules, we compared a number of B.subtilis expressing RNAi molecules in shrimp-WSSV mortality trials. We found that one of the tested strains (strain X) has a probiotic effect in Litopenaeus vannamei. We observed that feeding shrimp unmodified B.subtilis strain X resulted in increased resistance in shrimp to WSSV infection (35% reduction in mortality) and also to Vibrio parahaemolythicus infection (20% reduction in mortality). This strain was further modified to express a dsRNA targeting an essential WSSV gene, Vp19, or for expression dsRNA-Luc targeting a non-existent Luciferase gene as a dsRNA negative control. For virus challenge experiments, shrimp were fed strain X bacteria expressing dsRNA-Vp19, dsRNA-Luc or commercial food without bacteria for 5 days prior to WSSV challenge by injection.
Full protection (zero mortality) was observed at 10 days post-infection in shrimp receiving food top-coated with dsRNA-Vp19 expressing bacteria while the control group of shrimp fed commercial food had 85% mortality. Additionally, qPCR analysis of WSSV VP19 expression indicated a 3-log fold reduction in virus titer in surviving shrimp fed bacteria expressing dsRNA-vp19 compared to the control group. Using this Directed Biotics platform technology, we have achieved complete control of WSSV in the lab and are currently carrying out field trials.