System for the biocontrol of white spot syndrome virus (WSSV) in aquaculture
Inventors
Sayre, Richard T. • Vinogradova-Shah, Tatiana • Sineva, Elena
Assignees
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Publication Number
US-12144837-B2
Publication Date
2024-11-19
Expiration Date
Abstract
The inventive technology relates to novel paratransgenic strategies for the biocontrol of pathogens in animal systems using interfering RNA molecules expressed in genetically modified bacteria that may be configured to colonize a target host. In one preferred embodiment, the invention includes novel paratransgenic strategies for the biocontrol of pathogens in aquatic organisms raised in aquaculture environments.
Core Innovation
The invention relates to a paratransgenic dsRNA delivery system for biocontrol of white spot syndrome in shrimp using genetically modified bacteria. The bacteria express a heterologous nucleotide sequence encoding an inhibitory polynucleotide that downregulates expression of the vp19 gene of white spot syndrome virus (WSSV). The inhibitory polynucleotide comprises first and second inhibitory RNA polynucleotides that are complementary and form an inhibitory double stranded RNA molecule (dsRNA).
The inhibitory dsRNA is described in terms of RNA interference (RNAi) processing in the shrimp host, including processing of dsRNA by Dicer and loading into RISC, including Argonaute2, to generate siRNA. The dsRNA is engineered from nucleotide sequences having at least 95% homology to specified SEQ ID NOs 13 to 15 corresponding to WSSV vp19 and, optionally, vp28/Wsv477. The system achieves downregulation of vp19 expression in response to dsRNA delivery via engineered bacteria colonizing the gut.
The invention further includes delivery and persistence concepts in an aquaculture setting, including feed-mediated delivery and gut colonization by genetically modified enteric bacteria. The text additionally addresses transmission concepts including vertical transmission and horizontal transmission, and includes an optional gene switch for controlled expression. Experimental demonstrations described in the partial content include use of an RNase III-deficient Bacillus subtilis strain (BG322) delivering WSSV-specific dsRNA via feed, with reduced WSSV-associated mortality, reduced viral replication, and vp19-specific siRNA evidence by Northern blot.
Claims Coverage
The partial content identifies three independent claims covering compositions for biocontrol of white spot syndrome by using genetically modified bacteria to express inhibitory dsRNA that downregulates the WSSV vp19 gene. Across the independent claims, the inventive coverage is centered on dsRNA formation from complementary inhibitory RNA polynucleotides and homology-defined nucleotide sequences, with additional claim sets narrowing the bacterial and delivery contexts for shrimp biocontrol.
Inhibitory dsRNA vp19 downregulation by engineered bacteria
A composition for the biocontrol of white spot syndrome comprising a genetically modified bacteria expressing at least one heterologous nucleotide sequence having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 15, encoding at least one inhibitory polynucleotide that downregulates expression of the vp19 gene of WSSV, wherein said inhibitory polynucleotide comprises a first inhibitory RNA polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 13 and a second complementary polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 14, and wherein said first and said second RNA polynucleotides form an inhibitory double stranded RNA molecule (dsRNA) that downregulates expression of the vp19 gene of WSSV.
Biocontrol composition with complementary vp19 inhibitory polynucleotides
A composition for the biocontrol of white spot syndrome in shrimp comprising a genetically modified bacteria expressing a first inhibitory RNA polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 13 and a second complementary polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 14, wherein said first and said second RNA polynucleotides form an inhibitory dsRNA that downregulates expression of the vp19 gene of WSSV.
vp19 inhibitory dsRNA from SEQ ID NO. 13 and SEQ ID NO. 14
A composition for the biocontrol of white spot syndrome in shrimp comprising a first inhibitory RNA polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 13 and a second complementary polynucleotide having at least 95% homology with the nucleotide sequence according to SEQ ID NO. 14, wherein said first and said second RNA polynucleotides form an inhibitory dsRNA that downregulates expression of the vp19 gene of WSSV.
Collectively, the independent claims cover compositions where genetically modified bacteria express heterologous sequences encoding complementary inhibitory RNA polynucleotides, defined by at least 95% homology to SEQ ID NOs 13 and 14, that form inhibitory dsRNA. This inhibitory dsRNA downregulates WSSV vp19 gene expression, thereby supporting biocontrol of white spot syndrome in shrimp, with claim refinement in dependent claims addressing bacterial selection, feed-mediated administration, aquaculture context, and optional construct or cassette features.
Stated Advantages
Reduces WSSV-associated mortality in treated shrimp (reported ~50% reduction vs non-specific dsRNA in the partial content).
Lowers viral replication (reported 3–4 log reduction in the partial content).
Documented Applications
Biocontrol of white spot syndrome in shrimp using engineered bacteria expressing inhibitory dsRNA targeting the WSSV vp19 gene.
Feed-mediated delivery of the genetically modified bacteria or expressed dsRNA in shrimp aquaculture settings, including use with shrimp infected with or susceptible to WSSV.
Use of an RNase III-deficient genetically modified Bacillus subtilis strain (BG322) for delivery of WSSV-specific dsRNA with gut colonization concepts described in the partial content.
Detection of vp19-specific siRNA evidence (Northern blot) as a molecular readout in the described demonstrations.
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