Activation of the interfering RNA (RNAi) mechanism via genetically modified Bacillus subtilis against replication of the White Spot Syndrome virus (WSSV) in the shrimp Litopenaeus vannamei

Author: Jade Riet Graminho (Currículo Lattes)
Supervisor: Dr Luis Fernando Fernandes Marins
Co-supervisor: Dr Wilson Francisco Britto Wasielesky Junior

Abstract

Since 1992, the White Spot Syndrome Virus (WSSV) has been causing problems for the development of shrimp farming worldwide. Among the alternatives to control this virus is the use of double-stranded RNAs (dsRNAs) to block the expression of viral genes through the mechanism of RNA interference (RNAi) present in eukaryotes. The production and delivery of these molecules can be achieved in vitro through commercial kits or in vivo through gram-negative bacteria like Escherichia coli. The use of kits makes treatment more expensive and E. coli has a pathogenic potential. Thus, the use of probiotic bacteria as bioreactors of dsRNAs can be an interesting alternative. This dissertation aimed to validate the potential of a probiotic strain of Bacillus subtilis manipulated for the production of dsRNAs, in the protection of the shrimp Litopenaeus vannamei against WSSV. For this, the production of these molecules was optimized with the addition of nutrients to the culture media. The dsRNAs were extracted and purified from the probiotic and validated for their ability to activate the RNAi system in the hemocytes. In addition, the live probiotic was administered to the animals' diet and assessed for its ability to activate the RNAi system and also for its ability to protect shrimp against WSSV. The results showed that enrichment of the culture media significantly (p<0,05) increased the production of dsRNAs by the probiotic. Both the purified dsRNAs injected into the shrimp and the probiotic administered in the diet were able to activate the RNAi system, as evidenced by the increased expression of the related genes sid1 and argonaute2. Also, it was observed that the pretreatment for 15 days with the probiotic is sufficient to activate the RNAi system. When exposed to the virus, shrimp treated with the genetically modified probiotic had a significant (p<0,05) reduction in viral load, which resulted in a 34% survival. No viral inclusions were observed in the gills, cuticle and gastric epithelium of the survivors. This study demonstrates, for the first time, that a probiotic strain genetically modified to activate the RNAi system against a specific WSSV gene is capable of reducing viral load and increasing shrimp survival.

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